Generation of binding molecules

ABSTRACT

Provided are methods for efficiently and comprehensively screening antibody repertoires from B cells to obtain and produce molecules with binding characteristics and functional activities for use in human therapy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/821,502, filedNov. 22, 2017, now allowed, which is a continuation of U.S. Ser. No.14/856,417, filed Sep. 16, 2015, now issued as U.S. Pat. No. 9,908,946,issued Mar. 6, 2018, which is a continuation of U.S. Ser. No. 13/627,381filed Sep. 26, 2012, now issued as U.S. Pat. No. 9,145,588, issued Sep.29, 2015, which claims priority to U.S. Provisional Application No.61/539,116, entitled “Generation of Binding Molecules”, filed Sep. 26,2011, the entire contents of which are incorporated herein by thisreference.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 737572000203SeqList.txt,date recorded: Apr. 15, 2020, size: 30,801 bytes).

BACKGROUND OF THE INVENTION

The ability of the mammalian immune response to generate a large anddiverse antibody repertoire in response to antigen has been exploitedfor a range of applications in diagnostics, therapy and basic research.In particular, monoclonal antibodies, the products of a single B cellclone, have been broadly applied because of their well-definedspecificity and ease of production. Typically, monoclonal antibodies orthe genetic information encoding monoclonal antibodies with desirablespecificities are obtained from the B cells of animals or humans thathave been immunized with antigen or infected by pathogens.Alternatively, monoclonal antibodies may be obtained by one of severalrecombinant-DNA-based methods to construct and screen libraries ofantibodies or antibody fragments expressed on the surface ofbacteriophages or eukaryotic cells, or from in silica approaches.

Monoclonal antibodies have found increasing use in human therapy for thetreatment of a variety of diseases, including for example chronicinflammatory diseases and cancer. The immunogenicity of xenogeneicmonoclonal antibodies limits their use in the therapy of human disease.Exposure of patients to xenogeneic antibodies often results in adverseeffects or might lead to the neutralization and clearance of the appliedantibody, thereby reducing its pharmacological efficacy (Clark, 2000).Administration of humanized or fully human monoclonal antibodies topatients usually diminishes the aforementioned complications, inparticular when the amino acid sequences of the antibodies do notcontain epitopes that stimulate T cells. Antibodies encoded bynon-mutated, human germline heavy and light chain V gene segmentscontaining CDR3 regions devoid of T cell epitopes represent ultimateexamples of protein drugs with low immunogenicity (Ruuls et al., 2008;Harding et al., 2010). So, for therapeutic applications, monoclonalantibodies are preferably fully human, non-mutated and contain few or noT cell epitopes to prevent the formation of anti-drug antibodies.

B cells from the blood or lymphoid organs of humans have been used as asource of therapeutic monoclonal antibodies. Since the discovery of thehybridoma technology for immortalization of murine B cells (Kohler etal., 1975) and the realization that this technology could not be readilyreplicated using human B cells, several alternative methods for thegeneration of human monoclonal antibodies have been developed. Suchmethods include transformation of B cells through Epstein-Barr virusinfection (Tragiai et al., 2004), short term activation and expansion ofhuman B cells by stimulation with combinations of stimulator cells,antibodies and cytokines (Zubler 1987/Banchereau et al., 1991/Kim etal., 2001/Good et al., 2006/Ettinger et al., 2005) orretrovirus-mediated gene transfer (Kwakkenbos et al., 2010), cloning ofantibody V genes from single human B cells by PCR (Wrammert et al.,2010/Meijer et al., 2006), and identification and selection ofantigen-specific antibody-secreting B cells by hemolytic plaque assays(Babcook et al., 1996). Human B cell immortalization or activationtechniques are compatible with only some stages of B cell maturation andfurthermore, due to their low efficiencies (merely 1-3% of B cells) theyare not suitable for efficient interrogation of the whole repertoire ofspecific antibodies generated during a human immune response forantibodies with desired characteristics (Reddy et al., 2011).

Single-cell cloning, a procedure in which single human B cells areplated in microtiter well plates for analysis, has been used tocircumvent the low efficiencies associated with procedures that requireB cell activation and/or immortalization to obtain human monoclonalantibodies. In this approach, RNA from individual B cells is used toamplify the variable regions of the heavy and light chain (VH, VL) ofantibodies by PCR. The VH and VL genes are then inserted into suitableexpression vectors for transfection into cell lines and subsequentproduction of recombinant antibody fragments or full-length IgG (Smithet al., 2009/Tiller et al., 2008). Alternatively, amplified VH and VLgenes may be directly used for in vitro transcription and translation togenerate minute quantities of antibodies sufficient for binding analysisbut nor for assessing functional activity (Jiang et. al., 2006). Usingthese procedures, the production of recombinant monoclonal antibodies isnot limited to distinct B cell populations and does not depend on priorstimulation or immortalization. The major challenge in this approach isthe specific amplification of antibody genes by RT-PCR from single cellsand the occurrence of cross-contamination during handling of largenumbers of PCR reactions. Another practical limitation is the number ofindividual B cells that can be handled, which is typically restricted toseveral thousand, preventing extensive sampling of the entire antibodyrepertoire generated during an immune response. Finally, the method isrestricted to the analysis of readily accessible human B cells such asthose derived from blood and bone marrow.

Human monoclonal antibodies can also be isolated from recombinantantibody libraries in the laboratory, using one of the platforms forselection that in essence mimics the in vivo antibody response(Hoogenboom, 2005). For example, display technologies exploit largecollections of cloned antibody variable regions expressed on the surfaceof phage particles, bacteria, eukaryotic cells or ribosomes to selectfor antibodies that bind to antigens of interest (Ponsel et al.,2011/Clackson et al., 1991/Boder et al., 1997/Fuchs et al., 1991/Lee etal., 2007/Chao et al., 2006). The VH and VL regions inserted in thesedisplay systems are randomly combined to form collections of antibodybinding sites, i.e. fragments of intact IgG antibodies, which requirecorrect folding and assembly in e.g. prokaryotic cells for retrieval byantigen-binding methods. Display methods do not allow the retrieval ofantibodies from libraries through functional screening. In displayapproaches, original pairing of heavy and light chains is abrogated and,in addition, antibody-encoding DNA is lost as a result of the use ofrestriction enzymes during the cloning procedure. The success ofrecovering desired antibody specificities with in vitro antibodydiscovery techniques depends not only on the successful folding andexpression of the recombinant antibody fragments in e.g. prokaryoticcells but also on a range of screening parameters used during antibodyselection. These include the nature of the display platform, antigenconcentration, binding avidity during enrichment, the number ofselection rounds, and the design and diversity of the antibody libraries(Hoogenboom 2005/Cobaugh et al., 2008/Persson et al., 2006). Thus, dueto experimental procedures, folding requirements for expression ofantibody fragments in prokaryotic cells and parameters affecting thesuccess of antibody retrieval during selections, display systems do notpermit the comprehensive mining of antibody repertoires and do not allowdirect functional screening of human antibodies. Indeed,antigen-specific antibody fragments may be lost during subsequent roundsof antigen selection of phage display libraries (Ravn et al., 2010).

Transgenic mice harboring collections of human antibody genes have beenconstructed to alleviate some of the restrictions associated with theuse of human B cells as starting material for the generation of humanmonoclonal antibodies (Lonberg 2005). Such mice can be immunized withany antigen and their lymphoid organs are readily accessible forharvesting B cells. Once the transgenic mouse has been immunized,monoclonals can either be obtained through traditional hybridomageneration, by display technologies or using approaches that involve theharvesting, plating and screening of B cells, followed by isolation ofmAb genes and cloning into production cell lines.

For the generation of hybridomas, B cells from murine lymphoid organsare harvested and fused with myeloma cells to form immortalizedmonoclonal antibody-secreting cell lines. The low efficiency of cellfusion in hybridoma formation permits interrogation of only a fractionof the antibody repertoire and is restricted to B cell populations thatare amenable to fusion. If a satisfactory hybridoma is not formed, itbecomes difficult to obtain the antibody against challenging antigenssuch as membrane proteins. Thus, increasing the numbers of hybridomas isa crucially important step in screening the repertoire ofantigen-specific B cells from immunized mice and obtaining monoclonalantibodies with high affinity, specificity and desired functionalactivity (Kato et al., 2011/Li et. al., 1994; Crowe, 2009). In the mostefficient fusion protocols involving pre-stimulation of B cells andelectrofusion, approximately 1 in 1000 B cells fuses successfully with amyeloma cell to become an antibody-secreting hybridoma (Kato et. al.,2011). The hybridoma technology and other B cell immortalization methodsinterrogate the antibody-producing cells in pre-plasma cell B cellpopulations, specifically in memory B cells, or in circulatingshort-lived plasma blasts (Wrammert et al., 2008).

B cells from immunized transgenic mice with human antibody genes may beused to obtain collections of VH and VL regions that are randomlycombined to form combinatorial display libraries of human antibodyfragments. As argued above, due to experimental procedures, foldingrequirements for expression of antibody fragments in prokaryotic cellsand parameters affecting the success of antibody retrieval duringselections, display systems do not permit the comprehensive mining ofantibody repertoires and do not allow direct functional screening ofhuman monoclonal antibodies.

High-throughput sequencing has been utilized for sequencing of antibodyrepertoires derived from bone marrow plasma cells of protein-immunizedmice (Reddy et al., 2010). It was found that in the purified plasma cellpopulation, VH and VL repertoires were highly polarized with the mostabundant sequences representing 1-10% of the entire repertoire (Reddy etal., 2010). The most abundant VH and VL genes were randomly-paired,expressed as IgG molecules and screened for binding to the immunizingantigen.

A disadvantage of random pairing is that only 4% of the thus generatedantibodies were found to bind to the immunizing antigen. Theseantibodies had low affinities and/or poor expression levels andaggregation was frequently observed. The low proportion of specificantibodies could be improved by pairing VH and VL genes based on theirrelative frequency in the collection of sequences. In that case,following recombinant expression, approximately 75% of antibodies werefound to bind to antigen (Reddy et al., 2010). The disadvantage of VH/VLpairing according to relative frequencies is that collections of V-genesobtained by high throughput sequencing may contain VH and VL sequencesthat are present in similar frequencies yet are derived from different Bcell clones and thus may not represent a natural pair and may not form afunctional antibody molecule. Pairing of VH and VL regions based onfrequency is therefore inaccurate and may lead to the generation andscreening of many antibodies that have mismatched VH/VL pairs encodinglow affinity antibodies or antibodies that do not bind to the target ofinterest. Indeed, it has been shown that VH/VL pairing based on relativefrequencies yields a high proportion of modest to low affinityantibodies (Reddy et al., 2010). This implies that VH/VL pairing basedon high frequency of VH and VL genes present in large collections ofsequences is not predictive for the generation of high affinityantibodies. Thus, such an approach yields only small numbers VH/VLcombinations encoding antigen-specific antibodies which were generallyfound to have low affinities (Reddy et al., 2010).

A further disadvantage of the method reported by Reddy et al. is that itrelied on plasma cells as a source of antigen-specific monoclonalantibodies. Plasma cells represent only a small subpopulation ofB-lineage cells contributing to antibody diversity generated during animmune response. As a result antigen specific antibodies produced byother B cell populations during an immune response are not retrieved.These populations include short-lived plasma cells, transitional Bcells, germinal center B cells and IgM and IgG memory B cells present inlymphoid organs. When comparing antibody repertoires in these various Bcell populations, significant changes were observed (Wu, et al., 2010)which implies that a broader antibody repertoire is captured when more Bcell populations are included as source for VH/VL in deep sequencing.

Based on the above, it can be concluded that there is a need forantibody generation and selection approaches that facilitate theinterrogation of entire antibody repertoires for antibodies encoded byoriginal VH/VL pairs with desirable binding characteristics andfunctional activities.

SUMMARY OF THE INVENTION

To recapitulate, for selection and screening of the entire repertoire ofantibodies produced during an immune response it is necessary to exploittechniques that allow efficient retrieval of antigen-driven, clonallyexpanded B cells of various phenotypes and subpopulations and/or thegenetic information encoding the corresponding antibodies as originalVH/VL pairs. The present invention provides a method to efficiently andcomprehensively interrogate the broad spectrum of antibodies generatedby B cell populations. Preferably, these diverse B cell populations areobtained from transgenic animals, e.g. mice, which harbor human antibodygenes to facilitate immunization with any desirable antigen and generateantibodies for therapeutic application in humans. It is most preferredthat these transgenic mice have a limited VL repertoire, in particular asingle human rearranged VL. The method is independent of B cellimmortalization or activation procedures, facilitates screening ofantibody repertoires from B cells obtained from essentially all lymphoidorgans and does not require analyses of individual B cells. Preferably,to efficiently mine the entire repertoire of antibodies generated duringan immune response, B cells from every differentiation stage and lineageand present in relevant lymphoid organs such as lymph nodes, spleenblood and bone marrow are analyzed for the presence of monoclonalantibodies of desired specificity and characteristics such as affinityand functional activity. The method may thus address the entirepopulation of B cells in lymphoid organs or focus on B cellssubpopulations that are distinguishable based on phenotypiccharacteristics such as transitional B cells, memory B cells,short-lived plasma cells and the like. Furthermore, the method allowsdirect screening of antibodies for binding characteristics as well asfunctional activity in the relevant antibody format that isrepresentative for eventual application in human therapy. The inventionfurther provides for methods and means for production of these selectedantibodies in the desired formats as well as these antibodies and theiruses themselves. These uses include arrays as well as pharmaceuticalproducts.

The invention provides a method for producing a defined population ofbinding molecules, said method comprises at least the following steps:a) providing a population of B cells expressing a limited VL repertoirewherein essentially all of said B cells carry at least one VL, b)obtaining nucleic acids (RNA or DNA) from said B cells, c) optionally,amplification of nucleic acid sequences encoding immunoglobulin heavychain variable regions in said sample, d) at least partial sequencing ofall obtained nucleic acids of step b) or the amplification products ofstep c), e) performing a frequency analysis of sequences from step d),f) selecting desired VH sequences, g) providing a host cell with atleast one vector comprising at least one of said desired VH sequencesand/or at least one VL sequence of said limited VL repertoire, h)culturing said host cells and allowing for expression of VH and/or VLpolypeptides, i) obtaining said binding molecules. Alternatively, stepc) and d) can be replaced or supplemented by the alternative steps c′and d′: c′) constructing a cDNA library that is screened for VH regionspecific DNA sequences by probing with a nucleic acid probe specific forVH regions sequences and d′) at least partial sequencing of clonescontaining VH inserts. Where VH or VL is mentioned, functionalderivatives and/or fragments thereof are also envisaged.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the 4 mouse CH1 reverse primers and their position withinthe CH1 region are highlighted (1) Mouse CH1rev0; (2) CH1rev1; (3)CH1rev2; (4) CH1rev3. FIG. 1 discloses SEQ ID NOS 158 and 157,respectively, in order of appearance.

FIG. 2 depicts the VH amplification with forward primer DO_1177 andvarious CH1 reverse primers. Lane 1: DO_1171; Lane 2: DO_1172; Lane 3:DO_1173; Lane 4: DO_1174; Lane 5: negative control.

FIG. 3 is a graph showing the by-stander immune response against Fcportion of Fc-EGFR fusion protein upon immunization.

FIG. 4 depicts the ErbB2 specific IgG serum titer in ErbB2 vaccinatedmice.

FIG. 5 depicts the HA specific IgG serum titer in HA vaccinated mice.

FIG. 6 depicts the relative affinity of the anti-ErbB2 IgG polyclonalsera of ErbB2 vaccinated mice as determined by ELISA.

FIG. 7 is a graph showing the comparison of the percentages IgG B cellsper total iLN B cells per vaccination strategy and per antigen.

FIG. 8 shows a native SDS-PAGE analysis of a protein A purified singleVL human bispecific antibody preparation. MW: molecular weight. Lane 1,anti-thyroglobulin×anti-fibrinogen bispecific.

FIG. 9 depicts a native mass spectrometry analysis of a protein Apurified single VL human bispecific antibody for fibrinogen andthyroglobulin produced by co-transfection of HEK293 cells. The main peakof 144352 Da is the heterodimeric bispecific IgG species (96% of totalIgG); whereas the minor peaks of 144028 Da (3% of total IgG) and 142638Da (1% of total IgG) are the homodimeric parental IgG species.

FIG. 10 is a graph showing the potency of EGFR-specific mAb to rescueA431 cells from EGF-induced cell death.

DETAILED DESCRIPTION

The term ‘obtaining nucleic acids’ as used in step c) herein includesany methods for retrieving the sequence of nucleotides of nucleic acidsencoding VH sequences.

The term ‘defined population of binding molecules’ as used herein refersto at least two binding molecules that bind to at least one selectedantigen or epitope of interest. Preferably, the defined population ofbinding molecules comprises at least a significant portion, preferablyat least the majority and most preferably essentially all specificbinding molecules directed against the antigen or epitope of interestgenerated during an immune response. More preferably a population ofbinding molecules comprises between hundred and several thousand bindingmolecules, representing a significant portion of unique antigen-specificantibodies present in, e.g., a mouse immunized with said antigen. Thepopulation of binding molecules of the present invention may havedifferent specificities and/or affinities; i.e. may bind to differentepitopes of the selected antigen or may bind to the same epitope withdiffering affinities. The term ‘binding molecule’ as used herein means amolecule comprising a polypeptide containing one or more regions,preferably domains, which bind an epitope on an antigen. In a preferredembodiment, such domains are derived from an antibody.

The term ‘antibody’ as used herein means a protein containing one ormore domains that bind an epitope on an antigen, where such domains arederived from or share sequence homology with the variable region of anantibody. Antibodies are known in the art and include several isotypes,such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM. An antibodyaccording to the invention may be any of these isotypes, or a functionalderivative and/or fragment of these. Examples of antibodies according tothe invention include full length antibodies, antibody fragments,bispecific antibodies, immunoconjugates, and the like. Antibodyfragments include Fv, scFv, Fab, Fab′, F(ab′)₂ fragments, and the like.Antibodies according to the invention can be of any origin, includingmurine, of more than one origin, i.e. chimeric, humanized, or fullyhuman antibodies. Where the term functional fragment and/or derivativeis used in this specification it is intended to convey that at least oneof the functions, preferably the characterizing functions of theoriginal molecule are retained (in kind not necessarily in amount).Antibody binding is defined in terms of specificity and affinity. Thespecificity determines which antigen or epitope thereof is bound by thebinding domain. The affinity is a measure for the strength of binding toa particular antigen or epitope. Specific binding is defined as bindingwith affinities (K_(D)) of at least 1×10⁻⁵ M, more preferably 1×10⁻⁷ M,more preferably higher than 1×10⁻⁹ M. Typically, monoclonal antibodiesfor therapeutic application may have affinities of up to 1×10⁻¹⁰ M oreven higher.

The term ‘antigen’ as used herein means a substance or molecule that,when introduced into the body, triggers the production of an antibody bythe immune system. An antigen, among others, may be derived frompathogenic organisms, tumor cells or other aberrant cells, from haptens,or even from self structures. At the molecular level, an antigen ischaracterized by its ability to be “bound” by the antigen-binding siteof an antibody. In certain aspects of the present invention alsomixtures of antigens can be regarded as ‘antigen’. An antigen willcomprise at least one epitope. The term ‘epitope’ as used herein meansthe part of an antigen that is recognized by the immune system,specifically by antibodies, B cells, or T cells. Although epitopes areusually thought to be derived from non-self proteins, sequences derivedfrom the host that can be recognized are also classified asepitopes/antigens.

A ‘population of B cells’ as used herein may be any collection of Bcells. The B cells may be present as a subpopulation of other cells in asample. It may be derived from one or more individuals from the same ordifferent species. Preferably, the term ‘population of B cells’ means agroup of B cells obtained from at least one animal, preferably obtainedfrom lymphoid organs. In a preferred embodiment, a population of B cellscomprises essentially all splenic B cells. In a more preferredembodiment, the population further comprises also essentially all Bcells obtained from at least one lymph node. In a most preferredembodiment, the population of B cells comprises B cells obtained fromspleen, at least one lymph node, blood and/or bone marrow. In aparticularly preferred embodiment, a population of B cells comprisesessentially all B cells obtained from one or more lymphoid organs thatharbor the B cells that have clonally expanded as a result of antigenstimulation. Methods to obtain such B cell populations are known in theart. Of this population of B cells, essentially all B cells carry atleast one VL. In a preferred embodiment, essentially all B cells in thepopulation of B cells express a limited VL repertoire. In a mostpreferred embodiment, essentially all B cells in the population of Bcells carry the same VL.

The term ‘limited VL repertoire’ used herein means a restricted cohortof VL regions that supports the generation of a robust immune responseupon immunization and allows the efficient assembly of original VH/VLpairs with VH regions identified through the construction of heavy chainCDR3 heat maps. In one embodiment the limited VL repertoire comprises nomore than 100 different VL regions. When these 100 different VL regionswill be matched with a cohort of 100 different, grouped, VH regions itwill result in 10⁴ combinations (100 times 100) of VH/VL pairs that canstill be screened in functional assays. In a more preferred embodiment,the limited VL repertoire comprises no more than 10, more preferably nomore than 3 or 2 different VL regions, thereby further limiting thenumber of different VH/VL combinations and increasing the frequency oforiginal VH/VL pairs. In the most preferred embodiment, the limited VLrepertoire comprises no more than a single VL region. The advantage of asingle VL is that all antibodies that are generated upon encounter withan antigen share the same VL and are diversified only in the VH usage. AVL is defined by the particular combination of germline V and J genesegments and CDR3 region and includes somatically mutated variants ofsaid VL. Thus, the population of B cells expressing a limited VLrepertoire can mean that essentially no more than 100 VL, or preferablyless than 50 VL, or more preferably less than 10, or 3 or 2 VL or mostpreferably a single VL is expressed. In a preferred embodiment, all VLsin the limited VL repertoire are resistant to DNA rearrangements and/orsomatic hypermutations, preferably, the VL have a germline sequence. Thepreferred germline sequence is a light chain variable region that isfrequently used in the human repertoire and has superior ability to pairwith many different VH regions, and has good thermodynamic stability,yield and solubility. A most preferred germline light chain is 012,preferably the rearranged germline kappa light chainIgVκ1-39*01/IGJκ1*01 (nomenclature according to the IMGT database foundon the web at imgt.org) or fragment or a functional derivative thereof.Such single VL is also referred to as common VL, or common light chain.Obviously, those of skill in the art will recognize that “common” alsorefers to functional equivalents of the light chain of which the aminoacid sequence is not identical. Many variants of said light chain existwherein mutations (deletions, substitutions, additions) are present thatdo not materially influence the formation of functional binding regions.A “common light chain” according to the invention refers to light chainswhich may be identical or have some amino acid sequence differenceswhile retaining the binding specificity of the antibody. It is forinstance possible within the scope of the definition of common lightchains as used herein, to prepare or find light chains that are notidentical but still functionally equivalent, e.g. by introducing andtesting conservative amino acid changes, changes of amino acids inregions that do not or only partly contribute to binding specificitywhen paired with the heavy chain, and the like. It is an aspect of thepresent invention to use as single VL one identical light chain that cancombine with different heavy chains to form antibodies with functionalantigen binding domains (WO2004/009618, WO2009/157771, Merchant et al.,1998, Nissim et al., 1994).

The terms ‘amplification of nucleic acid sequences encodingimmunoglobulin heavy chain variable regions’ and ‘(at least partially)sequencing of nucleic acids’ have their usual meanings in the art.

The term ‘frequency analysis’ has its usual meaning in the art. For amore detailed explanation of the term, see e.g. example 1 of the presentinvention.

The term ‘desired VH sequences’ means those VH sequences that, based onthe frequency analysis, are produced in response to exposure to anantigen. These are assumed to encode VH regions that are specific forsaid antigen. Typically, an immune response to an antigen in a mouseentails the activation of about 100 different B cell clones (Poulsen etal., J Immunol 2011; 187; 4229-4235). Therefore, it is most preferred toselect the 100 most abundant clones. More practically, at least 20,preferably about 50 abundant clones are selected.

‘Host cells’ according to the invention may be any host cell capable ofexpressing recombinant DNA molecules, including bacteria, yeast, plantcells, eukaryotes with a preference for mammalian cells. Particularlywhen larger antibody formats are desired bacterial cells are notsuitable and mammalian cells are preferred. Suitable mammalian hostcells for expression of antibody molecules are known in the art.

It is an aspect of the invention to provide a method according to theinvention as described above, further comprising taking a sample of saidcultured cells, the sample comprising at least one of said bindingmolecules, and subjecting the samples to at least one functional assay,and selecting at least one cell that expresses a binding molecule withdesired characteristics.

A ‘functional assay’ as used herein means a test to establish propertiessuch as binding specificity, affinity, neutralizing activity, tumor cellkilling, proliferation inhibition or any other desired functionalcharacteristic or activity of the binding molecule produced according tothe methods of the invention. Such assays are used to determine early onwhether binding molecules obtained are suitable for the desired purpose.Said desired purpose may be a diagnostic and/or therapeutic application.In one embodiment of the invention, the method for producing a definedpopulation of binding molecules further comprises the step of harvestingthe supernatants of the cultured cells, the supernatants containing saidbinding molecules, and subjecting the supernatants to at least onefunctional assay. Irrespective of a functional assay as described above,the present invention also compasses ways to determine the identity ofthe binding molecules, using methods known in the art.

It is an aspect of the invention to provide a method according to theinvention as described above, further comprising providing said hostcell with means for expression of said at least one VH and VL in adesired format. The term ‘desired formats’ as used herein refers to aform of the binding molecule in which it can be used for its particularpurpose. The typical formats of binding molecules, in particularantibody like molecules that are suitable for particular purposes arewell known in the art. For therapy these molecules would typically befully human monoclonals (mono- or bispecific, and/or mixtures thereof,i.e. Oligoclonics®). For imaging these molecules would typically beantibody fragments, and so on. Desired formats include, but are notlimited to, bispecific formats such as DARTS™, BiTEs™, single lightchain bispecific antibodies (Merchant et al., 1998) includingCH3-engineered bispecifics such as knob-into-hole variants orcharge-engineered CH3 variants, DVD-Ig antibodies (\Vu et al., 2007),mixtures of antibodies (de Kruif et al., 2009) and the like.

In a further preferred embodiment, the population of B cells that isprovided for the method for producing a defined population of bindingmolecules is enriched for B cells that express immunoglobulin receptorsthat bind to the antigen. Such enrichment may occur when a B cellencounters antigen (e.g. when a mouse is immunized with an antigen) andis activated to divide to generate a clone of B cells. It is an objectof the present invention to provide a method for producing a populationof defined binding molecules further providing B cell clones whereinsaid collection of B cell clones comprise a collection of VH regionsthat are enriched for VH regions encoding antibodies directed to theantigen or epitope of interest. Such enrichment can, for example, becarried out by taking only those B cell clones obtained fromantigen-exposed animals as a starting population that are selectedthrough an antigen recognition process, i.e. by using selection methodscomprising coated or labeled antigen. Typically, the 20 most abundantclones, preferably, the 50 most abundant clones, more preferably the 100most abundant clones are selected; most preferably, the 200 mostabundant clones are selected.

In a preferred embodiment, the antigen-specific VH regions are clonallyrelated. In another preferred embodiment, the population of B cells ishighly-enriched for B cells that express immunoglobulin receptors thatbind to the antigen. In this case, the majority of B cells that areprovided will be antigen specific, and thus, an amplification step ofall nucleic acid sequences encoding VH regions may not be necessary andall isolated nucleic acids from said B cells can be, at least partially,sequenced directly. It is thus an aspect of the present invention, toprovide for a method for producing a defined population of bindingmolecules, said method comprising at least the step of providing apopulation of B cells expressing a limited VL repertoire wherein said Bcells comprise a collection of VH regions that is enriched for VHregions encoding antibodies directed to the antigen or epitope ofinterest.

It is an aspect of the invention to provide a method according to theinvention, wherein said population of B cells is obtained from atransgenic mouse carrying a limited, preferably human, VL repertoire.

It is an aspect of the invention to provide a method according to theinvention, wherein said mouse has been immunized such that selectiveclonal expansion of B cells that react with the antigen or epitope ofinterest is preferentially induced.

An ‘immunization protocol that causes the selective expansion of Bcells’ as used herein means that primary and booster immunizations aredesigned to cause selective expansions of B cells that produceantibodies that bind to the antigen or epitope of interest. Theimmunization protocol may for example use different forms or fragmentsof the antigen during primary immunization and each subsequent boosterimmunization. For example, the antigen may be expressed on the membraneof a cell, a recombinant protein, a recombinant protein fused to anotherprotein, a domain of a protein or a peptide of a protein. Theimmunization protocol may include the use of an adjuvant during theprimary and/or booster immunizations. In a preferred embodiment, anadjuvant is used during primary immunization only to limit the extent ofnon-specific expansion of bystander B cells. Bystander B cells are cellsthat are activated without the step of binding of antigen to theantibody receptor expressed on the surface of the B cell. It is known inthe art that immunization with Fc-fusion proteins for example, oftenresults in a robust anti-Fc response where up to about 70% of all Bcells react to the Fc part of the fusion protein rather than to theantigen of interest. In the most preferred embodiment, an immunizationprotocol is used without adjuvant to preferentially expand B cells thathave been activated by the antigen used for immunization. It istherefore an aspect of the invention to provide a method for producing adefined population of binding molecules, said method comprising at leastthe step of providing a population of B cells expressing a limited VLrepertoire, wherein said population of B cells is obtained from atransgenic mouse carrying a limited, preferably human, VL repertoire,wherein said mouse has been immunized with an antigen, such thatselective clonal expansion of B cells that react with the antigen orepitope of interest is preferentially induced. A preferred way ofinducing selective clonal expansion of B cells is DNA tattoovaccination. The term ‘DNA tattoo vaccination’ refers to an invasiveprocedure involving a solid vibrating needle loaded with plasmid DNAthat repeatedly punctures the skin, wounding both the epidermis and theupper dermis and causing cutaneous inflammation followed by healing(Bins 2005/Pokorna 2008).

In transgenic mice with human antibody genes, a plurality of human IgH Vregions and/or a plurality of human Ig light chain kappa V regions havebeen introduced in the genome of the animals (Lonberg 2005). Uponimmunization, these mice mount an antigen-specific immune response thatis diversified in heavy and light chain V region utilization. It isanticipated that, upon immunization of these transgenic mice withantigen, high throughput sequencing, frequency ranking of VH and VLgenes, construction of CDR3 heat maps and random or frequency-guidedpairing of VH and VL regions yields a large proportion of antibodiesthat do not bind to the antigen or bind with low affinity (Reddy et al.,2010). It is therefore an object of the present invention to usetransgenic animals that harbor a restricted repertoire of humanimmunoglobulin light chains for immunization purposes. Such transgenicanimals that harbor a limited repertoire of human light chains aredescribed in WO2009/157771. Preferably, the endogenous kappa light chainis functionally silenced in such animals to minimize the use of murinelight chains in antibodies generated in such mice. In a furtherembodiment, also the endogenous lambda light chain is functionallysilenced to further reduce the use of murine light chains in antibodies.In a most preferred embodiment, transgenic animals that carry a singlerearranged human VL region that is resistant to somatic hypermutation isused for immunization to generate antibodies in which the processes ofsomatic mutation and clonal expansion and selection mainly act on the VHregions of the antibody expressed on the membrane of a B cell. Hence,high throughput sequencing and creation of CDR3 heat maps to identifyantigen-driven, clonally expanded B cells and the antibodies they encodemay focus on VH regions only. It is therefore an aspect of the presentinvention to provide a method for producing a defined population ofbinding molecules, said method comprising the step of providing apopulation of B cells, wherein said population of B cells is obtainedfrom a transgenic animal, preferably a mouse, carrying a limited,preferably human, VL repertoire, wherein said animal has been immunizedwith an antigen. In a preferred embodiment, the invention provides for amethod for producing a defined population of binding molecules, saidmethod comprising the step of providing a population of B cells, whereinsaid population of B cells is obtained from a transgenic mouse carryinga single rearranged human VL, preferably the human IGVκ1-39 light chain(WO2009/157771). The advantage of this germline human IGVκ1-39 lightchain is its anticipated reduced immunogenicity due to absence of strongnon-self DRB1 binders (WO2009/157771, example 19). In addition, thislight chain is known to be capable of pairing with many different humanVH regions. Through an array of genetic mechanisms, the antibody VLrepertoire that can be generated in an animal is virtually unlimited.

In one aspect according to the invention, the method for producing adefined population of binding molecules further comprises the step ofproviding the host cell with means for expression of the at least one VHand VL in a desired format. The term ‘desired format’ as used hereinmeans that the selected VH and VL sequences are expressed together withother sequences such that antibody formats can be expressed within thehost cell. In one embodiment, after selection of suitable VHs, mixturesof antibodies are produced by a single cell by introducing at least 2different heavy chains and one common light chain into a cell(WO2004/009618). In another embodiment, after selection of suitable VHregions, the at least two different heavy chains are engineered suchthat heterodimerization of heavy chains is favored overhomodimerization. Alternatively, the engineering is such thathomodimerization is favored over heterodimerization. Examples of suchengineered heavy chains are for example the protuberance and cavity(knob-into-hole) constructs as described in WO98/050431, or thecharge-variants as described (Gunasekaran et al., 2010) orWO2009/089004, or WO2006/106905.

It is another aspect of the invention to provide a method for producinga defined population of binding molecules, wherein said bindingmolecules have a desired effect according to a functional screeningassay, the method further comprising the step of taking the supernatantsof said cultured cells, the supernatants comprising said bindingmolecules, subjecting the supernatants to at least one functionalscreening assay, and selecting at least one cell that expresses abinding molecule with desired characteristics. Preferably, said hostcell comprises a nucleic acid sequence encoding a common light chainthat is capable of pairing with said desired VH, such that producedantibodies comprise common light chains, as described above. In specificembodiments said culturing step and said screening step of the method isperformed with at least two clones. The method may optionally include anassay for measuring the expression levels of the antibodies that areproduced. Such assays are well known to the person skilled in the art,and include protein concentration assays, immunoglobulin specific assayssuch as ELISA, RIA, and the like.

The present invention inter alia describes a method for producing adefined population of binding molecules, wherein the starting point is apopulation of B cells that expresses a restricted repertoire of lightchain variable (VL) regions and a diversified repertoire of heavy chainvariable regions (VH). The VL region repertoire may for example berestricted by limiting the number of V and J genes available duringrecombination in a transgenic animal or by inserting one or a fewpre-rearranged VL regions in the genome of a transgenic animal, byreducing or abrogating the rate of somatic mutation occurring in the VLregion or by a combination of these strategies (WO2009/157771). The VHregions may be diversified by recombination of V, D and J gene segmentsand somatic mutation. Upon immunization, collections of heavy chainnucleic acid sequences are obtained from B cells in the lymphoid organsof transgenic animals, subjected to high throughput sequencing andanalyzed to rank all unique heavy chains based on frequency and to rankHCDR3 based on length, percentage identity and frequency to constructHCDR3 heat maps. Nucleotide sequence information is used to rank VHregions according to their frequency in the collection and frequentlyoccurring sequences, assumingly representing VH regions expressed in Bcells that have undergone clonal expansion as a result of antigenstimulation, are cloned into expression vectors in conjunction with oneof the VL regions present in the restricted repertoire. By using arestricted VL repertoire, the search for original VH/VL pairs is highlysimplified because no VL sequence information needs to be retrieved,analyzed or ranked from the immunized animal; in case the originalrestricted VL repertoire comprises a single VL, all VH/VL combinationswill represent original pairs as used by B cells in vivo. In case theoriginal restricted VL repertoire contained a few VL regions,combination with the ranked VH regions yields only small collections ofVH/VL combinations that can be rapidly screened for binding andfunctional activity.

Expression vectors containing the VH and VL regions are used totransfect cells to rapidly obtain antibodies for binding assays andfunctional screening. Different formats of antibodies can be obtained byusing expression vectors that contain different genetic elements that,for example, drive the formation of antibody fragments or antibodieswith different isotypes, bispecific antibodies, mixtures of antibodiesor antibodies that have engineered variable or constant regions formodified effector functions or modified half life, or contain additionalbinding sites, are devoid of amino acid sequences that have adeleterious effect on development, production or formulation ofantibodies such as glycoslylation and deamidation sites.

EXAMPLES Example 1: Deep Sequence Analysis and Frequency Ranking of VHGenes Expressed in Murine Spleen B Cells Using VH Family-SpecificPrimers

This example describes the use of high throughput sequencing to retrieveand analyze the repertoire of antibody VH regions expressed in thespleen of wild type mice immunized with the antigens ErbB2 or ErbB3.Because immunization will enrich the B cell population for clonesdirected against the immunogen, it is anticipated that sequencing largenumbers of VH transcripts identifies these B cell clones as they will bepresent within the population in higher frequencies. In this example,approximately 25,000 VH region genes from the spleen of a singleimmunized mouse are retrieved by high throughput sequencing and rankedbased on frequency.

Spleens were collected from mice immunized with either the antigen ErbB2or ErbB3 using DNA tattooing (see example 6). A single cell suspensionwas prepared according to standard techniques. B cells were isolatedfrom this splenic single cell suspension in a two-step MACS procedureusing materials from Miltenyi biotec(http://www.miltenyibiotec.com/en/default.aspx). Briefly, splenic Bcells were isolated by first depleting the non-B cells, followed bypositive selection of B cells. The non-B cells were depleted by labelingof T cells, NK cells, myeloid cells, plasma cells and erythrocytes witha cocktail of biotinylated antibodies (Table 1) and subsequentincubation with streptavidin Microbeads. Next the non-B cells weredepleted over an LD column. The flow through, containing the enriched Bcell fraction, was labeled with magnetic Microbeads using FITCconjugated anti-lgG1 and IgG2ab antibodies (Table 1) followed bylabeling with anti-FITC Microbeads (Miltenyi Biotec, Cat no.130-048-701). The IgG labeled cells were subsequently positivelyselected over an LS column (Miltenyi Biotec, Cat no 130-042-401). MACSprocedures were performed according to Kit/Microbead specific manualssupplied by Miltenyi. The purity of the isolated B cells was determinedby FACS analysis according to standard techniques using the antibodieslisted in Table 2.

TABLE 1 antibodies to label non-B cells Ab # Antigen Label CloneSupplier Cat no. Ab0064 IgG1 FITC A85-1 Becton Dickinson 553443 Ab0131IgG2ab FITC R2-40 Becton Dickinson 553399 Ab0158 CD138 Biotin 281-2Becton Dickinson 553713 Ab0160 CD3E Biotin 145-2C11 eBioscience 13-0031Ab0161 Ly-6G Biotin RB6-8C5 eBioscience 13-5921 Ab0162 TER-119 BiotinTER-119 eBioscience 13-5921 Ab0163 CD49b Biotin DX5 eBioscience 13-5971Ab0164 CD11b Biotin M1/70 eBioscience 13-0112

TABLE 2 antibodies for FACS analysis Ab # Antigen Label Clone SupplierCat no. Ab0064 IgG1 FITC A85-1 Becton 553443 Dickinson Ab0131 IgG2abFITC R2-40 Becton 553399 Dickinson Ab0067 CD138 APC 281-2 Becton 558626Dickinson Ab0160 IgM PE-CY7 II/41 eBioscience 25579082 Ab0161 IgD PE11-26 eBioscience 12599382 Ab0162 CD19 PerCP-cy5.5 1D3 eBioscience45019382 Ab0163 B220 Aallphyco- RA3-6B2 eBioscience 47045282 cyanin-efluor 780

To extract the nucleic acids of the B cells, cells were lysed in TrizolLS (Invitrogen). RNA was prepared and cDNA synthesized according tostandard techniques. Primers designed for amplification of murine VHrepertoires were taken as starting material and were modified for use in454 high throughput sequencing by addition of 454 primer sequences(forward 454 primer: CGTATCGCCTCCCTCGCGCCATCAG (SEQ ID NO: 1); reverse454 primer: CTATGCGCCTTGCCAGCCCGCTCAG) (SEQ ID NO: 2). The completeprimer sequences for the PCR amplification of murine VH repertoires areshown in Tables 3 and 4.

TABLE 3 The forward 454 Phusion primers, complete. Thephusion part (CGTATCGCCTCCCTCGCGCCATCAG) (SEQ ID NO: 1) isin italic, in bold is the 5′part of the VH genes. Name sequenceSEQ ID NO: wobble mIGHV1A_454 CGTATCGCCTCCCTCGC 3 K = 15% T/85% GGCCATCAG GAGKTCMAG M = 15% A/85% C CTGCAGCAGTC mIGHV1B_454CGTATCGCCTCCCTCGC 4 S1 = 5% G/95% C GCCATCAG SAGRTCCAS R = 5% A/95% GCTGCAGCAGTC S2 = 95% G/5% C mIGHV1C_454 CGTATCGCCTCCCTCGC 5S = 50% C/50% G GCCATCAG SAGGTCCAG H = 33% A/33% CTHCAGCAGTC C/33% TmIGHV1D_454 CGTATCGCCTCCCTCGC 6 S = 80% G/20% C GCCATCAG SAGRTCCAGR = 15% A/85% G CTGCAACAGTC mIGHV1E_454 CGTATCGCCTCCCTCGC 7K = 15% T/85% G GCCATCAG CAKGTCCAA CTGCAGCAGCC mIGHV1F_454CGTATCGCCTCCCTCGC 8 GCCATCAG CAGGCTTAT CTACAGCAGTC mIGHV1G_454CGTATCGCCTCCCTCGC 9 GCCATCAG CAGCGTGAG CTGCAGCAGTC mIGHV2_454CGTATCGCCTCCCTCGC 10 M = 15% A/85% C GCCATCAG CAGGTGCAG S = 50% C/50% GMTGAAGSAGTC m1GHV3_454 CGTATCGCCTCCCTCGC 11 S = 80% G/20% C GCCATCAGSAKRTGCAG K = 50% G/50% T CTTCAGGAGTC R = 15% A/85% G m1GHV4_454CGTATCGCCTCCCTCGC 12 GCCATCAGGAGGTGAAG CTTCTCCAGTC mIGHV5A_454CGTATCGCCTCCCTCGC 13 M = 15% A/85% C GCCATCAG GAAGTGMWG W = 80% A/20% TCTGGTGGAGTC mIGHV5B_454 CGTATCGCCTCCCTCGC 14 V = 20% C/40% GCCATCAGGAVGTGAAG G/40% A CTSGTGGAGTC S = 80% G/20% C mIGHV6A_454CGTATCGCCTCCCTCGC 15 R = 50% A/50% G GCCATCAG GAAGTGAAR M = 50% A/50% CMTTGAGGAGTC mIGHV6B_454 CGTATCGCCTCCCTCGC 16 GCCATCAG GATGTGAACCTGGAAGTGTC mIGHV6C_454 CGTATCGCCTCCCTCGC 17 GCCATCAG GAGGAGAAGCTGGATGAGTC mIGHV7_454 CGTATCGCCTCCCTCGC 18 M = 50% A/50% C GCCATCAGGAGGTGMAG R = 50% A/50% G CTGRTGGAATC mIGHV8_454 CGTATCGCCTCCCTCGC 19R = 50% A/50% G GCCATCAG CAGRTTACT W = 20% A/80% T CWGAAASAGTCS = 80% G/20% C mIGHV9_454 CGTATCGCCTCCCTCGC 20 S = 80% G/20% C GCCATCAGCAGATCCAG R = 15% A/50% G TTSGTRCAGTC mIGHV10_454 CGTATCGCCTCCCTCGC 21GCCATCAG GAGGTGCAG CTTGTTGAGTC mIGHV11_454 CGTATCGCCTCCCTCGC 22 GCCATCAGGAAGTGCAG CTGTTGGAGAC mIGHV13_454 CGTATCGCCTCCCTCGC 23 S = 50% C/50% GGCCATCAG SAGGTGCAG K = 50% G/50% T CTKGTAGAGAC mIGHV15_454CGTATCGCCTCCCTCGC 24 GCCATCAG CAGGTTCAC CTACAACAGTC

TABLE 4 The reverse 454 Phusion primers, complete. Thephusion part (CTATGCGCCTTGCCAGCCCGCTCAG) (SEQ ID NO: 2) is in italic.The part specific for the murine J segments are in bold. SEQ ID Namesequence NO: mIGHJ1_454 CTATGCGCCTTGCCAGCCCGCTCAG GAGG 25AGACGGTGACCGTGGTCCC mIGHJ2b_454 CTATGCGCCTTGCCAGCCCGCTCAG GAGG 26AGACTGTGAGAGTGGTGCC mIGHJ3_454 CTATGCGCCTTGCCAGCCCGCTCAG GCAG 27AGACAGTGACCAGAGTCCC mIGHJ4b_454 CTATGCGCCTTGCCAGCCCGCTCAG GAGG 28AGACGGTGACTGAGGTTCC

In the PCR, the four reverse JH primers were mixed in equal ratios priorto use. The forward primers were not mixed, so 22 PCR reactions wereperformed. The PCR reaction products were analyzed on gel and it wasexpected to yield PCR products of 350 to 400 base pairs in length. SomePCR products were mixed based on frequency of VH genes in normalrepertoires (Table 5, below). The intensities of the bands on gel wereexpected to correspond to the ratios listed in this table and when thiswas the case, PCR products were mixed for sequencing based on volumes.Where intensities of bands on gel did not correspond to ratios listed inTable 5, over- or under represented bands could be adjusted. As highthroughput sequencing also identifies the primer and the PCR reaction,ratios can always be ‘adjusted’ after sequencing and the frequency ofeach VH gene per PCR reaction analyzed.

TABLE 5 PCR products that can be pooled. Pool name Percentage (%) 1A 391B 1CD 1EFG 2 4 ¾ 7 5 15 6 1 7/8 10 9 16 10/11 6 13/15 2

Sequence analysis was performed in order to rank all unique VH genesfrom a single animal, immunization, and/or cell population on frequency:

-   -   Raw sequences were analyzed to identify those that encode a VH        region open reading frame that at least contains HCDR3 plus some        neighboring framework sequence to identify the VH. Preferably,        the VH region open reading frame contains CDR1 to framework 4    -   All sequences were translated into amino acid sequences    -   All sequences were clustered based on identical HCDR3 protein        sequence    -   All clusters were ranked based on number of VH sequences in each        cluster    -   An alignment of all sequences in each cluster was made based op        protein sequences, in which differences with the germline VH and        germline JH are indicated. All identical sequences in the        alignment are again clustered.

This provides information on the most frequently occurring VH genewithin a CDR3 cluster. This gene may have differences compared to thegermline as a result of somatic hypermutation. This gene is chosen forconstruction and expression with the common VL gene

High throughput sequencing was performed using Roche 454 sequencing onsamples from an individual immunized mouse. Other high throughputsequencing methods are available to a skilled person and are alsosuitable for application in the method. In total, 118,546 sequence readswere used as a raw data set to first identify sequences that representedfull length VH regions or portions thereof encoding at least 75 aminoacids. For each sequence within this set, frameworks 2-4 and all 3 CDRregions were identified as described (Al-Lazikani et. al., 1997).Sequences were subsequently subjected to a number of criteria includingthe presence of a canonical cysteine residue, the absence of stop codonsand the minimal length for each CDR regions. All VH regions fulfillingthese criteria were then clustered to identify the frequency in whicheach unique CDR3 are used, thereby generating heavy chain CDR3 heatmaps. Then, all identical clones in each CDR3 cluster were grouped andaligned with the germ line VH sequence. This analysis allows for theidentification of abundantly used VH genes in large repertoires.

Albeit that this analysis can be carried out manually, the use of analgorithm including the above instructions greatly facilitates theanalysis process (Reddy et al., 2010)

A total of 18,659 clusters were identified within 30,995 annotated VHsequences and 2,733 clusters were found that had more than 1 member. Inaddition, 123 clusters had more than 20 members. The first 40 clustersof these are shown in Table 6. Nine clusters had more than 100 members.The number of 30.000 sequences is more than sufficient as many clonesappear only once and the experiments are aimed at identifying frequentclones. In fact, less than 30.000 sequences would work quite well.Alignments of the two largest clusters demonstrated the presence of 100%germline genes and variants containing mutations throughout the VH gene(data not shown).

TABLE 6 Example of clusters identified by unique CDR3. SEQ # identicalID cluster # sequences HCDR3 NO: Cluster001 337 YSNYWYFDV 29 Cluster002212 GGLRGYFDV 30 Cluster003 130 YDSNYWYFDV 31 Cluster004 124 TYDNYGGWFAY32 Cluster005 116 AGLLGRWYFDV 33 Cluster006 116 RDY Cluster007 113RFGFPY 34 Cluster008 103 AITTVVATDY 35 Cluster009 102 AYYYGGDY 36Cluster010 99 SGPYYSIRYFDV 37 Cluster011 91 SEGSSNWYFDV 38 Cluster012 89GTLRWYFDV 39 Cluster013 76 DFYGSSYWYFDV 40 Cluster014 75 DNWDWYFDV 41Cluster015 73 FYDYALYFDV 42 Cluster016 72 GNYGSSYFDY 43 Cluster017 72WKVDYFDY 44 Cluster018 70 GGYWYFDV 45 Cluster019 69 YKSNYWYFDV 46Cluster020 66 LLPYWYFDV 47 Cluster021 64 SYYGSSYWYFDV 48 Cluster022 63GGYYGSRDFDY 49 Cluster023 63 DYDWYFDV 50 Cluster024 61 TYNNYGGWFAY 51Cluster025 57 GGLYYDYPFAY 52 Cluster026 57 WGDYDDPFDY 53 Cluster027 56DYYGSSYWYFDV 54 Cluster028 55 EATY 55 Cluster029 52 YGSSYWYFDV 56Cluster030 52 WGYGSKDAMDY 57 Cluster031 51 WGRELGNYFDY 58 Cluster032 49YGNYWYFDV 59 Cluster033 48 TVTTGIYYAMDY 60 Cluster034 48 HYYSNYVWWYFDV61 Cluster035 47 GALRGYFDV 62 Cluster036 47 HYYGSTWFAY 63 Cluster037 45LGAYGNFDY 64 Cluster038 44 REFAY 65 Cluster039 43 EAAYYFDY 66 Cluster04043 GSLRGYFDV 67

Example 2: Deep Sequence Analysis and Frequency Ranking of VH GenesExpressed in Murine IgG+ Spleen B Cells Using a Single Primer Set

In this example, a primer specific for the IgG CH1 constant region wasused to interrogate the repertoire of VH gene sequences expressed inIgG+ memory B cells in the spleen of mice immunized with the ErbB2-Fcfusion protein. At the 5′ end of the mRNA an oligonucleotide primer wasannealed to a triple guanine stretch that was added to each mRNA by anMMLV reverse transcriptase. This 5′ primer introduces a priming site atthe 5′ end of all cDNAs. Using this approach, amplifications of all VHregions expressed in IgG+ B cells can be done using the 5′ primer andthe CH1 primer, preventing a potential bias introduced by the use of alarge number of VH family-specific primers and focusing the analysis ona population of B cells that has apparently undergone activation andisotype switching as a result of stimulation with antigen.

Wild-type C57BL/6 mice were immunized intraperitoneally with ErbB2-Fcprotein (1129ER, R&D systems) dissolved in Titermax Gold adjuvant (TMG,Sigma Aldrich, T2684) on days 0, 14, and 28 and with ErbB2-Fc in PBS atday 42. Total splenic B cells were purified on day 45 by MACS procedureas detailed in Example 1. The total splenic B cell fraction fromsuccessfully-immunized mice, as determined by serum antibody titers inELISA, contained 5-10% IgG+ B cells. This material was used to optimizethe PCR conditions.

Transgenic mice containing the human HuVκ1-39 light chain, as describedin (WO2009/157771) were either immunized with EGFR-Fc fusion protein(R&D Systems, Cat no 334-ER) emulsified in Titermax Gold adjuvant (TMG,Sigma Aldrich, T2684) or, as a control, with Titermax Gold adjuvantemulsified with PBS at an interval of 14 days. The latter group wasincluded to identify the VH repertoire of B cells responding to theadjuvant alone. Mice were three times immunized with EGFR-Fc/adjuvantemulsion or adjuvant emulsion alone on days 0, 14 and 28. At day 35 theanti-EGFR serum titer was determined by FACS using standard procedures.Mice that had developed at day 35 a serum titer >1/1.000 received afinal intraperitoneal boost with EGFR-Fc protein dissolved in PBS at day42 followed by collection of spleen at day 45 for isolation of splenic Bcells. Splenic B cells were isolated from the total spleen by positiveselection using mouse CD19-specific magnetic beads. The splenic B cellfraction was lysed in Trizol LS to isolate total RNA.

After RNA isolation cDNA was prepared (cell populations from HuVκ1-39light chain transgenic mice and mock and control/test sample withsimilar cell population) using MMLV reverse transcriptase in thepresence of primers mouse CH1 rev 0, 1, 2 or 3 together with MMLV 454 fw(Table 7). Four different primers were tested to identify the one thatresulted in optimal cDNA yields and PCR products; MMLV was designed tocontain the 3′ GGG stretch similar to the Clontech primers, deletingcloning sites but adding 454 sequences (SMARTer™ RACE cDNA AmplificationKit, Cat #634924, Clontech).

As a control, cDNA was also prepared using the standard Clontechprotocol; the SMARTer PCR cDNA synthesis kit using the SMARTer II Aoligo together with the gene specific CH1 rev 0, 1, 2 or 3 primers(SMARTer™ RACE cDNA Amplification Kit, Cat #634924, Clontech). Usingstandard procedures, the optimal PCR cycle number and optimal primercombination were determined. The Clontech protocol was followed for PCRconditions.

Next, material from immunized HuVκ1-39 light chain transgenic mice andmock-immunized mice was amplified under optimal conditions. Preferablyamplifications were carried out with a reverse primer upstream from theprimer used in cDNA synthesis to obtain a more specific PCR product(nested PCR). cDNA was cloned in pJET according to the manufacturer'sinstructions (CloneJET PCR cloning kit, Fermentas #K1232) andSanger-sequence 100 clones. PCR product of material derived from theimmunized and mock-immunized animals was purified and used for 454sequencing. The data were analyzed and used to construct CDR3 heat mapsare constructed as described in example 1.

TABLE 7 Primers used in this study. Primer MMLV 454 fwhybridizes at the 5′end of the mRNA. The position of the CH1 rev primersis indicated in FIG. 1. SEQ ID number Name Sequence NO: DO_1165 MMLV 454

 

68 fw GGG DO_1166 454 fw

 

1 DO_1167 mouse CH1 TGATGGGGGTGTTGTTTTGG 69 rev 0 DO_1168 mouse CH1CAGGGGCCAGTGGATAGAC 70 rev 1 DO_1169 mouse CH1 GCCCTTGACCAGGCATCC 71rev 2 DO_1170 mouse CH1 CTGGACAGGGATCCAGAGTTC 72 rev 3 DO_1171 mouse CH1CTATGCGCCTTGCCAGCCCGCTCAG T 73 454 rev 0 GATGGGGGTGTTGTTTTGG DO_1172mouse CH1 CTATGCGCCTTGCCAGCCCGCTCAG C 74 454 rev 1 AGGGGCCAGTGGATAGACDO_1173 mouse CH1 CTATGCGCCTTGCCAGCCCGCTCAG G 75 454 rev 2CCCTTGACCAGGCATCC DO_1174 mouse CH1 CTATGCGCCTTGCCAGCCCGCTCAG C 76454 rev 3 TGGACAGGGATCCAGAGTTC DO_1175 smart IVAAGCAGTGGTATCAACGCAGAGTGGC 77 oligo CATTACGGCGGG DO_1176 smart IVAAGCAGTGGTATCAACGCAGAGTGGG 78 oligo short DO_1177 5PCR

 

79 primer AAGCAGTGGTATCAACGCAGAGT 454 Legends: 454 5′ sequence

 

 (SEQ ID NO: 1) 454 3′ sequence CTATGCGCCTTGCCAGCCCGCTCAG (SEQ ID NO: 2)SMART sequence AAGCAGTGGTATCAACGCAGAGT (SEQ ID NO: 80) SfiIGGCCATTACGGCC (SEQ ID NO: 81)

The results of these experiments yield heavy chain CDR3 heat maps thatrepresent sequences used by B cells that have undergone activation andisotype switching as a result of stimulation by antigen. The VH regionsequences present in the most frequently-occurring clusters derived fromthe transgenic mice containing the human IGVκ1-39 light chain can beused to combine with the sequence of human IGVκ1-39 light chain tocollections of human monoclonal antibodies enriched for antibodiesspecific for EGFR.

Example 3: Deep Sequence Analysis and Frequency Ranking of VH GenesExpressed in Murine IgG+ Spleen B Cells without VH Family SpecificPrimers

For this example, the objective was to optimize deep sequencingtechnology of IgG VH genes by using amplifications based on primers thatamplify all Ig heavy chains and thus to prevent potential biasintroduced by VH family specific primers.

Antibody VH gene amplification for phage display library generation usesprimers that append restriction sites for cloning to VH genes at therequired position within the genes. This requires primer annealing siteswithin the VH genes and therefore VH family specific primers. In example1 such primer sequences were used. A downside of the use of VH familyprimers is that they amplify a subset of all VH genes in the repertoire.As a large collection of primers and PCR reactions is used to amplifyall genes and the PCR products are mixed afterwards, this will result inskewing of the ratios of the VH genes originally present in the samplesand therefore over/under representation of VH genes in the finalsequenced repertoire. To circumvent these problems in this study theRACE (Rapid Amplification of cDNA Ends) amplification protocol was usedin combination with an IgG1-CH1 specific primer set. The SMARTer RACEkit (Clontech; cat #634923 & #634924) was used which couples a 5′synthetic adaptor to the mRNA. The SMARTScribe RT enzyme produces a copyof the RNA transcript. The SMARTScribe RT starts the cDNA synthesis fromthe IgG mRNA at an anti-sense primer that recognizes a known sequence inthe IgG mRNA such as the poly A tail in this study. When the SMARTScribeRT enzyme reaches the 5′end of the IgG RNA template it adds 3 to 5residues to the 3′ end of the first-strand cDNA. The SMARTer oligo IIAcontains a terminal stretch of modified bases that anneals to thisextended tail added by the SMARTScribe allowing the oligo to serve as atemplate for the RT. Subsequently the SMARTScribe RT switches templatesfrom the mRNA molecule to the SMARTer oligo IIA, generating a completedouble strand cDNA copy of the original RNA with the additional SMARTersequence at the end. Thereafter, for PCR amplification of VH cDNA, anoligo that anneals to the 5′ SMART sequence on one end of the cDNA (5PCRprimer 454) and an IgG-CH1 specific primer at the other end of thecDNA are applied (Table 8). In this way the VH regions from IgG heavychains are amplified with just one primer combination, independent fromVH family specific primers, and in this case a primer specific for theIgG CH1 constant region was used to interrogate the repertoire of VHgene sequences expressed in IgG+ memory B cells in the spleen of miceimmunized with the ErbB2-Fc fusion protein. In the best case the IgG-CH1specific reverse primer should anneal as close as possible to the VHgene to increase the chance that the full VH gene can be sequenced.

Samples for deep sequencing were obtained from two immunized transgenicmice carrying the human huVκ1-39 light chain; mice were numbered mouse1145 and mouse 1146. Briefly, transgenic mice containing the humanHuVκ1-39 light chain, as described in (WO2009/157771) were immunizedwith EGFR-Fc fusion protein (R&D Systems, Cat no 334-ER) emulsified inTitermax Gold adjuvant (TMG, Sigma Aldrich, T2684). Mice were threetimes immunized with EGFR-Fc/adjuvant emulsion on days 0, 14 and 28. Atday 35 the anti-EGFR serum titer was determined by FACS using standardprocedures. Mice that had developed at day 35 a serum titer >1/1.000received a final intraperitoneal boost with EGFR-Fc protein dissolved inPBS at day 42 followed by collection of spleen at day 45 for isolationof splenic B cells. Splenic B cells were isolated from the total spleenby positive selection using mouse CD19-specific magnetic beads. Thesplenic B cell fraction was Iysed in Trizol LS to isolate total RNAaccording to standard procedures.

cDNA was synthesized from these RNA samples using the SMARTer RACE cDNAAmplification kit according to manufacturer's instructions to come toso-called RACE-Ready cDNA. This RACE-ready cDNA was subsequentlyamplified by PCR according to manufacturer's instructions and the SMARTspecific primer (Table 8) and one of several IgG CH1 specific primerswere used to establish which IgG-CH1 specific primer amplifies the IgGtranscript best. The IgG-CH1 specific reverse primers DO_1171 to DO_1174containing the 3′ 454 sequences were tested and as a forward primer theSMART tag specific primer containing the 5′ Roche 454 sequencing tag(DO_1177) was used (see table 8 and FIG. 1) (SMARTer™ RACE cDNAAmplification Kit, Cat #634923 & 634924, Clontech). The PCR scheduleused is shown in Table 9.

TABLE 8 Primers used in this study. The position of theCH1 rev primers is indicated in FIG 1. SEQ ID number Name Sequence NO:DO_1171 mouse CH1 CTATGCGCCTTGCCAGCCCGCTCAG TG 73 454 rev 0ATGGGGGTGTTGTTTTGG DO_1172 mouse CH1 CTATGCGCCTTGCCAGCCCGCTCAG CA 74454 rev 1 GGGGCCAGTGGATAGAC DO_1173 mouse CH1 CTATGCGCCTTGCCAGCCCGCTCAGG 75 454 rev 2 CCCTTGACCAGGCATCC DO_1174 mouse CH1CTATGCGCCTTGCCAGCCCGCTCAG C 76 454 rev 3 TGGACAGGGATCCAGAGTTC DO_11775PCR

79 primer 454 AAGCAGTGGTATCAACGCAGAGT Legends: 454 5′ sequence

 

 (SEQ ID NO: 1) 454 3′ sequence CTATGCGCCTTGCCAGCCCGCTCAG (SEQ ID NO: 2)SMART sequence AAGCAGTGGTATCAACGCAGAGT (SEQ ID NO: 80)

TABLE 9 PCR schedule. Step Temperature Time Number of cycles 1 98° C. 30seconds 1 2 98° C. 25 seconds 10 3 72° C.-54° C. 25 seconds Touchdown 472° C. 50 seconds 5 98° C. 25 seconds 14 6 58° C. 25 seconds 8 72° C. 50seconds 9 72° C.  3 minutes 1 10 16° C. ∞

PCR results are shown in FIG. 2 demonstrating that DO_1171 did not givea PCR product, whereas the other three all gave an abundant PCR product,see FIG. 2. It was concluded that DO_1172 is the best option to amplifythe VH region, since this primer is closest to the VH gene and producesin PCR a clear and specific band.

The PCR was performed several times on template (Table 9) to obtainenough material (500-1000 ng) for deep sequencing. To determine the DNAconcentration after PCR, a fluorimetric quantification of DNA wasperformed. In this case, Quant-IT Picogreen dsDNA (Invitrogen P7589)measurements were performed using a Biotek Synergy. PCR product ofmaterial derived from the immunized animals (samples 1145 and 1146) wassubsequently sent to Eurofins MWG Operon (found on the web ateurofinsdna.com) for high throughput sequencing.

Eurofins first ligated barcoded linkers to each of the two samples. Inthis way both samples could be sequenced in one chip-segment, therebyreducing costs. With this layout Eurofins provided sequencing with GSFLX+ technology where length read is 600-700 bp on average.

Deep sequencing revealed more than 50.000 reads per mouse and data wereanalyzed as explained in example 1. Table 10 provides the 25 largestclusters from two of the analyzed samples.

TABLE 10 Cluster size, HCDR3 sequence and VH genes usedare presented from cDNA samples from mouse1145 and 1146. #/25 provides the frequencyof the VH gene in 25 clusters. sample SEQ 1145 Cluster ID Name SizeCDR3 sequence NO: VH gene #/25 Cluster001 1243 HYSDYPYFDY 82 J558.66.16519 Cluster002 858 YGDYINNVDY 83 J558.66.165 Cluster003 543 GFYGYDF 847183.19.36 1 Cluster004 376 LDTIVEDWYL 85 J558.66.165 DV Cluster005 335LDTVVEDWYF 86 J558.66.165 DV Cluster006 320 YGDYSNYVDY 87 J558.66.165Cluster007 313 TRQFRLRDFD 88 J558.83.189 1 Y Cluster008 290 FDYGSTQDYA89 J558.66.165 MDY Cluster009 213 SGNYDFYPMD 90 J558.66.165 Y Cluster010205 RLVEY 91 J558.66.166 1 Cluster011 197 YGDYSNNVDY 92 J558.66.165Cluster012 194 LDDGYPWFAY 93 J558.55.149 1 Cluster013 182 LSDYGSSAYL 94J558.66.165 YLDV Cluster014 180 QVDYYGSSYW 95 J558.66.165 YFDVCluster015 179 LGYGSSYLYF 96 J558.66.165 DV Cluster016 175 LGYGSIYLYF 97J558.66.165 DV Cluster017 168 LTDYGSGTYW 98 J558.66.165 FFDV Cluster018162 LDYYGSSYGW 99 J558.66.165 YFDV Cluster019 162 YGDYINSVDY 100J558.66.165 Cluster020 159 YTDYINSVDY 101 J558.66.165 Cluster021 156LDTIVEDWYF 102 J558.66.165 DV Cluster022 148 DYYGSSYGFD 103 VGAM66.165 1Y Cluster023 147 IYSNSLIMDY 104 J558.66.165 Cluster024 143 LGYGSSYWYF105 J558.66.165 DV Cluster025 142 GGYYPYAMDY 106 J558.12.102 1 Total7190 # different VH 7 sample SEQ 1146 ID Name Size CDR3 NO: VH #/25Cluster001 2570 EGRGNYPFDY 107 36-60.6.70 2 Cluster002 1791 DYSYYAMDY108 J559.12.162 1 Cluster003 1251 MRLYYGIDSS 109 3609.7.153 3 YWYFDVCluster004 905 MRLFYGSRYS 110 3609.7.153 YWYFDV Cluster005 841SYYYGSRESD 111 J558.53.146 1 Y Cluster006 614 GKYYPYYFDY 112 J558.12.1022 Cluster007 515 WGSSGY 113 J558.55.149 1 Cluster008 477 TGYNNYGSRF 114J558.18.108 3 IY Cluster009 441 RLVDY 115 J558.67.166 3 Cluster010 378WWFLRGVYVM 116 J558.85.191 4 DY Cluster011 306 TGYNNYGSRF 117J558.18.108 TY Cluster012 303 RLVEY 91 J558.67.166 Cluster013 291 RLIEY118 J558.67.166 Cluster014 291 GDWYFDV 119 VGAM.8-3-61 2 Cluster015 265RQFLLGVYAM 120 J558.85.191 DY Cluster016 237 RHFLLGVYAM 121 J558.85.191DY Cluster017 230 EGRVTTLDY 122 36-60.6.70 Cluster018 216 GDWYFDY 123VGAM.8-3-61 Cluster019 212 MRLFYGSSYS 124 3609.7.153 YWYFDV Cluster020203 GSGYVYAMDY 125 VGAM3.8-4-71 1 Cluster021 200 GTTAYYAMDY 126VGAM3.8-3-61 2 Cluster022 197 TGYNNYGSRF 127 J558.18.108 AY Cluster023182 GKYYPYYFVY 128 J558.12.102 Cluster024 163 GTTSYYAMDY 129VGAM3.8-3-61 Cluster025 153 RGSYGTCFDY 130 J558.85.191 Total 13232# different VH 12

The results from Table 10 show that different VH genes were amplifiedand sequenced in the PCR and deep sequencing procedures, Sample 1145contains many clusters within the 25 largest clusters that use theJ558,66.165 gene. Sample 1146 contains a large diversity of VH geneswith 12 different VH genes in which each is present between one and fourtimes within the 25 largest clusters. These results suggest that themethod allows unbiased amplification and analysis of IgG VH repertoires.

To conclude, these experiments resulted in a ranking of the mostfrequent VHs that represent sequences used by B cells that haveundergone activation and isotype switching as a result of stimulation byantigen. The VH region sequences present in the mostfrequently-occurring clusters derived from the transgenic micecontaining the human IGVκ1-39 light chain can be used to combine withthe sequence of human IGVκ1-39 light chain to collections of humanmonoclonal antibodies enriched for antibodies specific for EGFR.

Example 4: Immunization Strategies for the Construction of Reliable VHCDR3 Heat Maps

A broad array of Immunization methods is available that use variousformats of antigen in combination with adjuvant to optimize theantigen-specific immune response in animals. For the ranking offrequently used heavy chain genes optimally representing VH regions fromB cells that have expanded as a result of stimulation with the antigenof interest, it is critical that immunization protocols are used thatfocus the immune response on said antigen or even on an epitope of saidantigen. Thus, the use of antigens fused or coupled to carrier proteins(such as Fc fusion proteins or proteins coupled to carriers like KeyholeLimpet Hemocyanine, known in the art) is to be avoided or restricted toa single step in the immunization procedure like a single primaryimmunization or a single booster immunization. It is expected that evenlimited activation of B cells through the use of carrier or fusionproteins or adjuvant may show up in ranked VH sequences/HCDR3 heat maps,thereby contaminating the analysis. Ideally, the immunizations are thusperformed with ‘essentially pure antigens’. The present exampledemonstrates that single or repeated immunization with an antigen fusedto an Fc-portion indeed results in expansion of irrelevant B cells, i.e.B cells that react with the Fc-portion rather than with the antigen ofinterest.

Single human VL transgenic mice (group 1) and wildtype mice (group 2)were immunized with the EGFR overexpressing tumor cell line A431 on days0 and day 14 (2×10E6 A431 cells in 200 μl PBS), followed by ipimmunization with Fc-EGFR fusion protein emulsified in Titermax Gold. Atday 35, serum was collected and tested in ELISA for the presence ofanti-Fc antibodies.

As control, a third group of mice comprising both single human VLtransgenic mice and wildtype mice (group 3) were immunized with Fc-EGFRfusion protein only on days 0, 14, 28, 42 and 52. At day 56, serum wascollected and tested in ELISA for the presence of anti-Fc antibodies.

The results (FIG. 3) show that even after a single immunization withFc-EGFR, antibodies against the Fc portion are detected in groups 1 and2. Although the levels of anti-Fc antibodies in these two groups werelower than the levels of anti-Fc as observed in group 3, the VH regionsencoding these anti-Fc antibodies will be found in CDR3 heat maps,constructed as described in example 1. If so desired, such Fc-specificbinders can either be circumvented by using essentially pure antigenssuch as in DNA tattoo or by elimination of Fc-specific binders from thepopulation of B cells prior to VH sequence analysis.

Example 5: DNA Vaccination by Tattooing

DNA vaccination exploits plasmid DNA encoding a protein antigen toinduce an immune response against said protein antigen. There is no needfor purification of proteins for immunization and proteins, includingmembrane proteins, are expressed in their natural configuration on acell membrane (Jechlinger et al., 2006/Quaak, et aI., 2008/Stevenson etal., 2004).

In this example, we have used DNA tattoo vaccination, an invasiveprocedure involving a solid vibrating needle loaded with plasmid DNAthat repeatedly punctures the skin, wounding both the epidermis and theupper dermis and causing cutaneous inflammation followed by healing(Bins 2005/Pokorna 2008). Here we used DNA tattoo vaccination strategiesto induce antibody responses in mice. The goal was to assess the qualityof the antibody response in the absence or presence of adjuvant. Asdescribed in example 4, for the construction of adequate CDR3 heat maps,it is desirable to focus the antibody response on the antigen ofinterest, omitting the use of adjuvant that causes clonal expansion ofunwanted B cells.

For tattoo vaccination, plasmids encoding human ErbB2 and plasmidsencoding influenza virus Hemagglutinin (HA) were used. Three DNA tattoovaccination strategies were tested to optimize the priming and boostingof the immune response: (A) vaccination with vector DNA encoding theErbB2 or HA antigen, (B) vaccination of vector DNA encoding ErbB2 or HAtogether with an adjuvant or (C) heterologous prime-boost vaccinationwith DNA encoding ERbB2 or HA followed by a boost with purified ErbB2 orHA protein in TM Gold adjuvant. In addition, control group (group D)mice were immunized with purified ErbB2 or HA in TM Gold adjuvant. Toestablish an optimized DNA tattoo vaccination protocol the followingimmunization protocols were used:

Group A (DNA only): In this group, mice were vaccinated on day 0, 3, and6 with plasmid DNA encoding ErbB2 or HA in the absence of adjuvantfollowed by a boost with the same DNA after four weeks. No adjuvant wasused.

Group B (DNA+ genetic adjuvant): To test if an adjuvant increases thepriming of the humoral immune response, plasmid DNA encodingTANK-binding kinase 1 (TBK1) was co-vaccinated with ErbB2 or HA plasmidDNA. It has been shown that TBK1 acts as an adjuvant for DNA vaccinationusing a gene gun (Ishii et al., 2009). Comparison of group B with groupA will reveal what impact the genetic adjuvant has on the generation ofantibodies specific for HA or ErB2. Animals in group B were DNAvaccinated at same time points as those in group A. To examine thecontribution of genetic adjuvant in priming of the immune system,plasmid DNA encoding TBK-1 was mixed in a 1:1 ratio with pVAX1-ErbB2 orpVAXI-HA and subsequently administrated by DNA tattoo. To this end miceare vaccinated with 20 μg pTBK-1 and 20 μg pVAX1-ErbB2 or pVAX1-HA in 10μl PBS.

Group C (DNA+protein): In this group a heterologous prime-boost protocolwith DNA tattoo followed by intraperitoneal (i.p.) protein boost wastested to examine if a final protein boost is required to induce anantigen-specific serum IgG titer of > 1/1000 and if this boost isnecessary to efficiently induce splenic memory B cells. I.p. injectionis the direct immunization route to the spleen. So, by first priming theimmune system by DNA vaccination ErbB2 or HA is presented to the immunesystem as in vivo expressed protein. Subsequently, the primed immunesystem was boosted with ErbB2 or HA in adjuvant via the i.p. injectionroute to induce a systemic immune reaction. Comparison of group C to Areveals the impact of the systemic boost on 1) antigen-specific IgGserum titer and 2) on generation of the splenic memory B cellcompartment.

Immunization and first boost with pVAXI-ErbB2 or pVAX1-HA were carriedout according to the scheme described for group A. Subsequently mice areboosted at day 28 and day 42 with 20 μg of protein in 200 μl emulsion ofTitermaxGold adjuvant or in 200 μl PBS, respectively, administrated viaip injection. For HA vaccination mice were injected with 20 μg HA(Meridian life Science Inc, Cat no R01249). For ErbB2 mice were injectedwith 20 μg of a truncated ErbB2 protein: the extra cellular domain (ECD,aa23-652) of ErbB2 fused to FC-tail (R&D systems, Cat no 1129ER).

Group D (protein): In this control group mice were vaccinated i.p. withErbB2 or HA in adjuvant. Material from this group served as positivecontrol for the analyses of the samples from groups A-C. At day 0, 14and 28 mice were vaccinated with 20 μg of ErbB2 or HA in 200 μl emulsionof TitermaxGold adjuvant. For final boost, mice received 20 μg of ErbB2or HA dissolved in 200 μl PBS.

Serum titer and affinity were determined after each boost by ELISA andFACS respectively using standard protocols (Middendorp et al., 2002). Tostudy affinity maturation during the vaccination protocol, the relativeaffinity of the polyclonal antigen-specific-lgG serum was determined byELISA. The efficacy and quality of memory B cell induction at the end ofeach vaccination strategy was examined by FACS in spleen and draininginguinal lymph node (iLN) as described (Middendorp et al., 2002).

All ErbB2 DNA immunized mice developed an anti-ErbB2 IgG serumtiter >10,000 after two immunization rounds (FIG. 4A-C), indicating thattwo rounds of DNA immunization via tattoo are sufficient to induce astrong anti-ErbB2 antibody response. The results show protein immunizedmice develop a strong anti-ERbB2 IgG serum titer at day 21 (FIG. 4D).

A third immunization round at day 28 with DNA (groups A1 and B1)resulted in a further increase of the anti-ErbB2 IgG serum titer at day35 (seven days after third vaccination round) and day 45 (end point)(FIGS. 4A and 3B). In group B1 we found that co-administration of theErbB2 expression vector (group A1) together with a DNA-adjuvant (thepBoost3 vector encoding the TBK1 protein), would boost the antibodyresponse against ErbB2. Comparison of the anti-ErbB2 serum titer in timeshowed that the mice in the groups A1 and B1 developed a comparableanti-ErbB2 serum titer (FIG. 4E-G). This indicated that for the ErbB2antigen co-administration of the pBoost3 vector failed to enhance thepolyclonal anti-ErbB2 IgG serum titer. The mice in group C1 firstreceived two immunization rounds (day 0 and 14) with DNA followed by aboost (at day 28) with ErbB2-Fc protein emulsified with TitermaxGoldadjuvant. This protein boost at day 28 resulted in a strong increase ofthe anti-ErbB2 IgG serum titer at day 35 (after third immunization atday 28) and day 45 (end point) (FIG. 4C). At day 35 the serum titer ofgroup C1 (DNA-protein) was higher compared to the DNA only vaccinatedmice (groups A1 and B1) and marginal lower compared to protein onlyimmunized mice (group D1). At day 45 the serum titers of the groups C1(DNA and protein) and D1 (protein only) were comparable.

All mice that were immunized with the HA antigen via the fourvaccination strategies developed a strong anti-HA IgG serum titer at day21 (data not shown) and 35 (FIG. 5). The anti-HA IgG serum titer betweenthe DNA (group A2) and DNA+adjuvant (group B2) strategies werecomparable. Moreover, DNA vaccination followed by a boost with HAprotein emulsified with Titermax Gold (group C2) or three timesimmunization with protein (group 02) gave higher serum titer than DNAonly vaccination. In summary, the similarities and differences betweenthe four mice groups vaccinated with HA antigen were comparable to theresults observed for the ErbB2 vaccinated mice in terms of antigenspecific IgG serum titer.

To further compare the vaccination strategies we compared the polyclonalanti-ErbB2 IgG serum based on relative affinity. The relative affinitywas measured in day 45 sera samples, obtained three days after the finalboost. The relative affinity was determined by ELISA by incubating at afixed serum dilution on an ErbB2 antigen titration starting at 0.5μg/ml. The selected fixed serum dilution was based on the serum dilutionat which the sera reached the plateau using a fixed concentration ofErbB2 antigen in ELISA (0.5 μg/ml). The fixed serum dilution was 1:1,500for groups A1 and B1, and 1:20,000 for the groups C1 and D1. To comparethe individual groups we calculated and plotted the relative bindingbased on the reduction of absorbance versus the antigen dilution range.For each antigen concentration we calculated the relative binding, theOD of 0.5 μg/ml was set to 100%.

First we compared DNA vaccination with (group B1) and without DNAadjuvant (group A1) (FIG. 6A). No difference was observed in therelative binding between the groups that received DNA (group A1) orDNA+adjuvant (group B1). This indicated that the DNA adjuvant did notenhance the affinity of the polyclonal serum. In addition, to comparethe contribution of priming of the immune response with DNA followed bya protein boost we compared the sera of DNA-protein (group C1) andprotein (group D1) (FIG. 6B). The relative binding was significanthigher for group C1 than for group D1 (p<0,001 at antigen concentrations0.0625 and 0.0313 μg/ml). This suggested that the relative affinity ofthe polyclonal serum was on average higher for mice in group C1 than formice in group D1.

Isolation and analysis of tissues from immunized mice: Total splenic andtotal inguinal lymph node fractions from ErbB2 and HA vaccinated micewere collected and saved in Trizol LS. The draining inguinal lymph nodefrom the tattooed leg was isolated and saved in Trizol LS. In additionwe enriched the splenic IgG B cell fraction by MACS from mice immunizedusing strategy C1 and C2. Finally we determined the fraction of splenicIgG+ B cells in all ErbB2 and HA vaccinated mice. To isolate the splenicIgG+ B cells we performed a two step MACS purification. In the firststep we depleted the non-B cell using biotinylated non-B cell specificantibodies. In the second step we enriched the splenic IgG B cells usinganti-lgG1 and anti-lgG2ab specific antibodies. Table 11 gives anoverview from which mice the splenic IgG (lgG1 and IgG2ab) B cells wereisolated. Purity of the isolated IgG fractions was determined by FACSusing B cell specific and IgG1/lgG2ab specific antibodies. The % Ig Bcells was determined by staining a fraction of cells alter the depletionstep. Table 11 summarizes the yield and purity of the isolated IgG+ Bcell fractions per mouse and antigen used for immunization.

TABLE 11 yield and purity of the isolated IgG+ B cell fractions Strategyand % B % IgG B Total experimental Animal cells/total cells/B IgG cellsgroup Antigen number life gate cell gate (E + 0.6) DNA and protein ErbB231 68.46 30.27 1.36 DNA and protein ErbB2 32 67.37 39.96 1.76 DNA andprotein ErbB2 33 20.78 61.75 3.63 DNA and protein ErbB2 34 72.97 73.671.17 DNA and protein ErbB2 35 86.67 77.34 ND DNA and protein ErbB2 3684.98 82.12 ND DNA and protein ErbB2 37 85.03 74.32 ND DNA and proteinHA 38 90.41 55.48 2.09 DNA and protein HA 40 92.60 49.09 1.17 DNA andprotein HA 41 86.03 47.02 0.84 DNA and protein HA 42 88.46 46.99 1.21

To examine what protocol gives the best induction of the memory B cellcompartment, we examined the size of IgG B cell compartment in thespleen and the iLN per each vaccination strategy by FACS. We used acocktail of anti-lgG1-FITC and anti-lgG2ab-FITC monoclonals to visualizethe IgG B cell fraction. Table 12 gives an overview of the percentages Bcells within the lymphocyte gate and the percentages IgG B cells withinthe B cell fraction per mouse and tissue.

TABLE 12 IgG+ B cell fraction in the spleen and lymph node of ErbB2vaccinated mice Spleen iLN % B % IgG+ B % B % IgG+ B cells/ cells/cells/ cells/ Animal lympho. lympho. lympho. lympho. Group number gategate gate gate DNA (A1) 1 57.50 1.27 58.26 1.86 DNA (A1) 2 51.81 1.0951.09 2.80 DNA (A1) 3 54.05 1.32 58.93 2.43 DNA (A1) 4 54.06 1.73 55.242.93 DNA (A1) 5 54.93 2.72 53.78 6.45 DNA (A1) 6 58.68 1.98 53.89 2.90DNA (A1) 7 56.46 5.92 52.01 10.74 DNA + Adju (B1) 17 49.05 4.33 51.047.02 DNA + Adju (B1) 17 55.84 1.55 53.80 4.38 DNA + Adju (B1) 18 44.134.05 54.78 6.31 DNA + Adju (B1) 19 34.99 5.56 53.39 15.47 DNA + Adju(B1) 20 51.81 1.41 60.30 2.40 DNA + Adju (B1) 21 54.13 1.21 56.35 2.84DNA + Adju (B1) 22 58.34 2.11 62.29 1.80 DNA + protein 31 48.99 6.2654.12 6.49 (C1) DNA + protein 32 49.76 7.38 51.79 7.67 (C1) DNA +protein 33 45.94 12.76 45.14 4.19 (C1) DNA + protein 34 30.50 16.0744.83 10.91 (C1) DNA + protein 35 43.48 26.62 44.60 8.16 (C1) DNA +protein 36 41.12 24.07 45.13 11.00 (C1) DNA + protein 37 30.50 21.1030.82 9.17 (C1)

The mice from group C1 (DNA-protein) that received a correct i.p. boostwith ErbB2-Fc protein at day 28 (mice 35-37) had the largest fraction ofIgG+ B cells per splenic B cell population. This was expected as i.p.injection is the direct route of the antigen to the spleen. Thepercentages of IgG+ B cells in the iLN of group A1 (DNA) and B1(DNA+adjuvant) ranged between 1.8-15.47 with an averaged IgG+ B cellfraction of 4.3 and 5.7 for group A1 or B1, respectively. Interestingly,the mice from group A1 and B1 with the highest fraction of IgG+ B cellsin the iLN also contained a higher percentage of splenic IgG+ B cells.Analysis of the percentages of IgG B cells in the iLN of the HAvaccinated mice showed that these mice had on average comparablepercentages of IgG+ B cells per all iLN B cells as found in the A1 andB1 groups. The averaged percentages of IgG+ B cells in the iLN of DNAvaccinated (group A2) and the DNA+adjuvant vaccinated (group B2) micewas 5.0 and 3.0, respectively. FIG. 7 gives a comparison of thepercentages IgG B cells/iLN B cells per vaccination strategy and perantigen. In summary, these data showed that the draining iLN of DNA only(group A) of DNA+adjuvant (group B) vaccinated mice had a significantfraction of IgG+ B cells. Mice that received a protein boost (group C)contained a larger IgG+ B cell population in spleen and iLN.

We conclude that mice that were vaccinated with DNA or vaccinated withDNA and boosted with protein developed a strong antigen specific IgGserum titer. The relative affinity of the sera against ErbB2 can besignificantly enhanced using a DNA+protein immunization protocol insteadof a protein immunization protocol. The small variation of the antigenIgG serum titer between individual DNA vaccinated mice shows that theDNA tattoo method has been carried out consistently. DNA vaccinationwith ERbB2 and HA both resulted in strong antigen specific antibodyresponse.

It was reported that the adjuvant effect of plasmid DNA is mediated byits double-stranded structure, which activates Tbkl-dependent innateimmune signaling pathways in the absence of HRs (Ishii et al., 2008).Therefore, co-administration of a Tbkl-expressing plasmid was expectedto further boost DNA vaccine-induced immunogenicity. In our setting, wedid not observe a beneficial effect of the co-administration of the Tbklencoding pBoost3 vector. Co-administration of pBoost3 together with theantigen encoding vector failed to result in a higher serum titer,increased relative affinity or enhanced IgG B cell formation.

FACS analysis showed that the draining ilN in a mouse vaccinated withonly DNA (group A and B) contains a significant IgG+ B cell fraction.However the number of IgG B cells that can be isolated from a singledraining ilN is very limited due to the size of an ilN. Moreover, thefraction of IgG+ B cells in the ilN varied significantly betweenindividual DNA vaccinated mice. This could be the results of variationin administration of DNA via DNA tattoo. Interestingly, the mice with ahigh percentage of ilN IgG+ B cells also had a higher percentage ofsplenic IgG+ B cells. Another strategy to obtain a larger number of IgG+B cells is to boost the DNA vaccinated mice once with protein emulsifiedin TitermaxGold. The mice that were vaccinated with DNA and protein(group C) developed a significant splenic IgG+ B cell fraction (inaddition to a large ilN IgG+ B cell fraction). If no protein isavailable to for boost immunizations, mice can be boosted with cellsexpressing the antigen or virus-like particles expressing the antigen.

In conclusion, DNA vaccination via DNA tattooing is an effective androbust vaccination strategy to induce an antigen specific humoral immuneresponse. The mice that were vaccinated with only DNA (group A)developed a detectable IgG+ B cell fraction.

Example 6: Construction of Eukaryotic Vectors for the EfficientProduction of Single VL Bispecific Human Monoclonal Antibodies

One aspect of the present invention concerns the possibility of usingsequence information from VH gene frequency analysis and/or HCDR3 heatmaps to generate panels of antibodies in a desirable therapeutic formatand screen those antibodies for binding and or functional activity. Onesuch format is a bispecific IgG molecule. Conventional IgG molecules arecomprised of two identical heavy- and two identical light chains. Heavychains are polypeptides made up from separate domains: a VH region forantigen recognition, the CH1 domain, the hinge region, the CH2 domainand the CH3 domain. Pairing of the heavy chains to form a homodimer isthe result of high affinity interactions between the CH3 domains, whereafter covalent coupling of the two heavy chains results from disulfidebridge formation between cysteines in the hinge region of the heavychains.

The CH3 region has been used to introduce amino acid substitutions thatinhibit the formation of homodimers (pairing of heavy chains with anidentical CH3 region) while promoting heterodimerization (pairing of 2different heavy chains with complementary, engineered CH3 regions). Thishas resulted in efficient heterodimer formation upon co-expression ofCH3 engineered heavy chains (Gunasekaran et al., 2010; WO2006/106905;WO2009/089004).

In this example, we have constructed and tested expression vectors forthe efficient production of human bispecific single VL antibodies. Theoverall strategy is that genetic constructs encoding 2 differentantibodies are co-transfected into a single cell. By using complementaryengineered CH3 regions for the 2 different heavy chains, formation ofheterodimers (bispecific antibodies) is favored over the formation ofhomodimers (monospecific antibodies). Previously, it was shown that thecombination of K409D:K392D in the CH3 domain of one heavy chain incombination with D399′K:E356′K in the CH3 domain of the second chain(thus, so-called complementary engineered CH3 regions) drives theheterodimerization of human heavy chain constant regions in anengineered bispecific molecule (Gunasekaran 2010). We used to the sameamino acid pairs in the CH3 regions of constructs encoding single VL IgGantibodies to establish whether this would result in the efficientproduction of bispecific single VL human IgG monoclonal antibodiesthrough heterodimerization.

The rearranged single IGVκ1-39 VL gene was cloned into a eukaryoticexpression vector that contains the human gamma1 and kappa constantregions essentially as described (Throsby et al., 2008/de Kruif et al.,2009). Vector MV1201 contains DNA encoding a CH3 domain with theK409D:K392D amino acid substitutions in combination with a VH regionencoding a human single VL monoclonal antibody specific for fibrinogen.Vector MV1200 contains DNA encoding a CH3 domain with the D399′K:E356′Kamino acid substitutions in combination with a VH region encoding ahuman single VL monoclonal antibody specific for thyroblobulin(Gunasekaran et al., 2010/de Kruif et al., 2009).

MV1201 and MV 1200 were co-transfected into HEK293T cells andtransiently expressed as described (de Kruif et al., 2009). After 13days, supernatants were harvested and purified by protein A affinitychromatography using standard protocols. The protein A-purified IgG wasanalyzed by SDS-PAGE under reduced and non-reducing conditions; stainingof proteins in the gel was carried out with colloidal blue. The resultsof this experiment are shown in FIG. 8 Under non-reducing SDS-PAGE, asingle band with molecular weight of 150 kD was detected, showing thatwith these constructs hetero- and or homodimers were formed and no IgGhalf molecules consisting of a non-paired heavy/light chain combination.

After protein A purification, mass spectrometry was used to identify thedifferent IgG species produced by the transiently transfected cells. Asshown in FIG. 9, the supernatant contained 96% heterodimeric IgG and 3%and 1% of each of the parental monoclonal antibodies. Thus, theseprotein A supernatants can be immediately used for screening of bindingactivity and/or functional assays of bispecific antibodies.

Example 7: Generation of Bispecific Antibodies Specific for ErbB1 andErbB2 Tumor Antigens and Analysis of Tumor Cell Killing and Inhibitionof Tumor Cell Proliferation

Erbb1 and ErbB2 are growth factor receptors that play an important rolein tumor development and progression. Combinations of monoclonalantibodies against ErbB1 and ErbB2 have shown synergistic effects inanimal models of cancer (Larbouret et al., 2007) and therefore representpromising therapeutics for the treatment of cancer in humans. In thisexample, we demonstrate that, upon immunization, human monoclonalantibodies can be obtained from transgenic mice with a single humanlight chain through high throughput sequencing and creation of CDR3 heatmaps and that combinations of ErbB1 and ErbB2 antibodies with additiveand/or synergistic effect can be rapidly identified by in vitroscreening.

Transgenic mice with a single human light chain are immunized with ErbB2DNA and protein as described in example 5. Using the same protocols,another group of mice is immunized with ErbB1 DNA and proteins using thesame procedures. For ErbB1 and ErbB2 immunized mice, spleens areisolated and the VH repertoire of IgG+ B cells is analyzed by highthroughput sequencing as described in examples 1 and 2. Afterconstruction of CDR3 heat maps, the top 100 VH sequence groups for eachErbB1 and ErbB2 immunized mice are selected for further analysis. Forthe collection of ErbB1 and ErbB2 VH sequences, VH sequencesrepresentative for each cluster, this is the VH gene that is presentmost frequently within a cluster, which may be a germline gene or a genecontaining mutations, are cloned in an expression vector containing thesingle light chain and the CH3 mutation; ErbB1 VHs in MV1200 and ErbB2VHs in MV1201. HEK293 cells are transiently transfected with all 2500combinations (50 times 50) of cloned ErbB1 and ErbB2 VH sequence groupsusing the expression constructs that drive heterodimerization to formErbB1×ErbB2 bispecific antibodies as described in example 6. After 13days, culture supernatants are harvested and purified using protein Aaffinity chromatography. Purified IgG is used in functional assays oftumor cell killing and inhibition of tumor cell proliferation known inthe art.

After identification of ErbB1×ErbB2 bispecific antibodies with potentanti-tumor activity, VH sequence collections present in the clustersthat have an identical or similar CDR3 and that are used in thefunctional bispecific antibodies can be further deconvoluted using thesame approach to find those VH members in the collection that give themost potent anti-tumor activity.

Example 8: Deep Sequencing and Diversity Analysis of HCDR3 from SamplesObtained from Splenic B Cells from Non-Immunized Versus Immunized Mice

To demonstrate that the selective expansion of clones identified byunique HCDR3 sequences upon immunization can be analyzed by deepsequencing, splenic B cells from non-immunized and immunized mice weresubmitted to enrichment for B cells as described above. Nucleic acidswere isolated and, where needed amplified as described above and cDNAwas sent to Eurofins for high throughput sequencing.

Mice transgenic for huVκ1-39 and for a human heavy chain (HC) minilocuswere immunized with protein only (fused to Fc) or by using alternatingprotein and cellular immunizations (with cells expressing the sameantigen on their surface). cMet and EGFR were used as antigens in thisexample (two animals per group):

-   -   Group A: cMet-Fc in Titermax Gold adjuvant (TMG) on days 0, 14        and 28, and cMet-Fc in PBS on day 47.    -   Group B: cMet-Fc in TMG on days 0, 14 and 28, MKN45 cells in PBS        on day 49, and cMet-Fc in PBS on day 64.    -   Group C: EGFR-Fc in TMG on days 0, 14 and 28, and EGFR-Fc in PBS        on day 54.    -   Group D: EGFR-Fc in TMG on days 0, 14 and 28, A431 cells in PBS        on day 49, and EGFR-Fc in PBS on day 64.

Doses used were 20 μg protein in 125 μl TMG, 20 μg protein in 200 μl PBSand 2×10E6 cells in 200 μl PBS. Non-immunized transgenic mice werearound the same age as immunized mice at sacrifice (16 weeks, three micein total).

Spleens were collected from all mice (for immunized mice three daysafter the last immunization) and processed as described in Example 2. Tobe able to sequence many different samples in a mixture at reduced cost,a material identification (MID) tag specific for each mouse was added atthe 5′ end of each primer used in PCR amplification of cDNA togetherwith the SMART sequence (as detailed in Example 2). MID tag additionthus allowed pooling of material from several mice after PCRamplification and before 454-sequencing. Primers for amplification werethus adjusted to include MID tag complementary sequences (Table 3).

For non-immunized mice, spleen cell suspensions were enriched forB-cells using anti-CD19 Microbeads (Miltenyi Biotec, cat. no.130-052-201) and then sorted by flow cytometry to isolate mature,antigen-naive B cells expressing IgM or IgD. This was done by sortingfor CD19-positive, B220-positive, huVκ1-39-positive and mouse lightchain-negative B-cells and discriminating these in IgM-positive orIgD-positive cell fractions. In reverse primers used for cDNA synthesis,sequences were used that annealed to either IgM or IgD coding sequences.To be able to perform pooled sequencing for different samples, MID tagswere used here to identify material from different samples (Table 14).

TABLE 13 Primers used for deep sequencing material fromimmunized mice (one MID tag per mouse). MID SEQ ID Primer Primer tagSequence* NO: type name MID-01 ACGAGTGCGT AAGCAGT 131 Forward SMART-GGTATCAACGCAGAGT MID1-fw MID-01 ACGAGTGCGTCAGGGGC 132 Reverse mIgG-CH1-CAGTGGATAGAC MID1-rev MID-02 ACGCTCGACA AAGCAGT 133 Forward SMART-GGTATCAACGCAGAGT MID2-fw MID-02 ACGCTCGACACAGGGGC 134 Reverse mIgG-CH1-CAGTGGATAGAC MID2-rev MID-03 AGACGCACTC AAGCAGT 135 Forward SMART-GGTATCAACGCAGAGT MID3-fw MID-03 AGACGCACTCCAGGGGC 136 Reverse mIgG-CH1-CAGTGGATAGAC MID3-rev MID-04 AGCACTGTAG AAGCAGT 137 Forward SMART-GGTATCAACGCAGAGT MID4-fw MID-04 AGCACTGTAGCAGGGGC 138 Reverse mIgG-CH1-CAGTGGATAGAC MID4-rev MID-05 ATCAGACACG AAGCAGT 139 Forward SMART-GGTATCAACGCAGAGT MID5-fw MID-05 ATCAGACACGCAGGGGC 140 Reverse mIgG-CH1-CAGTGGATAGAC MID-rev MID-06 ATATCGCGAG AAGCAGT 141 Forward SMART-GGTATCAACGCAGAGT MID6-fw MID-06 ATATCGCGAGCAGGGGC 142 Reverse mIgG-CH1-CAGTGGATAGAC MID6-rev MID-07 CGTGTCTCTA AAGCAGT 143 Forward SMART-GGTATCAACGCAGAGT MID7-fw MID-07 CGTGTCTCTACAGGGGC 144 Reverse mIgG-CH1-CAGTGGATAGAC MID7-rev MID-08 CTCGCGTGTC AAGCAGT 145 Forward SMART-GGTATCAACGCAGAGT MID7-fw MID-08 CTCGCGTGTCCAGGGGC 146 Reverse mIgG-CH1-CAGTGGATAGAC MID8-rev *The MID tag sequence is underlined; the SMARTsequence is in bold; the IgG constant HC sequence in regular type.

TABLE 14 Primers used for deep sequencing material fromnon-immunized mice (one MID tag per cell population per mouse). Cell SEQfrac- MID ID Primer Primer tion* tag Sequence** NO: type name lgM^(HIGH)MID-04 AGCACTGTAG AAGCAGT 137 Forward SMART- B cells GGTATCAACGCAGAGTMID4- mouse 1 fw lgM^(HIGH) MID-04 AGCACTGTAGGGCCACC 147 Reverse mouseB cells AGATTCTTATCAGAC IgM- mouse 1 CH1- MID4- rev lgD^(HIGH) MID-05ATCAGACACG AAGCAGT 139 Forward SMART- B cells GGTATCAACGCAGAGT MID5-mouse 1 fw lgD^(HIGH) MID-05 ATCAGACACGCAGTTCT 148 Reverse mouse B cellsGAGGCCAGCACAGTG IgD- mouse 1 CH1- MID5- rev lgM^(HIGH) MID-10 TCTCTATGCGAAGCAGT 149 Forward SMART- B cells GGTATCAACGCAGAGT MID10- mouse 2 fwlgM^(HIGH) MID-10 TCTCTATGCGGGCCACC 150 Reverse mouse B cellsAGATTCTTATCAGAC IgM- mouse 2 CH1- MID10- rev lgD^(HIGH) MID-11TGATACGTCT AAGCAGT 151 Forward SMART- B cells GGTATCAACGCAGAGT M D11-mouse 2 fw lgD^(HIGH) MID-11 TGATACGTCTCAGTTCT 152 Reverse mouse B cellsGAGGCCAGCACAGTG IgD- mouse 2 CH1- MID11- rev lgM^(HIGH) MID-16TCACGTACTA AAGCAGT 153 Forward SMART- B cells GGTATCAACGCAGAGT MID16-mouse 3 fw lgM^(HIGH) MID-16 TCACGTACTAGGCCACC 154 Reverse mouse B cellsAGATTCTTATCAGAC IgM- mouse 3 CH1- MID16- rev lgD^(HIGH) MID-17CGTCTAGTAC AAGCAGT 155 Forward SMART- B cells GGTATCAACGCAGAGT MID17-mouse 3 fw lgD^(HIGH) MID-17 CGTCTAGTACCAGTTCT 156 Reverse mouse B cellsGAGGCCAGCACAGTG gD- mouse 3 CH1- MID17- rev *B cells selected forexpression of only huVκ1-39 LC. **The MID tag sequence is underlined;the SMART sequence is in bold; the IgD or IgM constant sequences inregular type.

For analysis of the sequencing results, custom designed algorithms wereused for VH gene identification and alignment of HCDR3 regions. Briefly,raw sequence data were imported into a dedicated computer program, whichtranslated all nucleotide sequences into six potential protein readingframes, each of which was then submitted to the following sequentialfilter criteria to find correct and complete human VH genes:

-   -   Sequences shorter than 75 amino acids were rejected as this was        considered as the minimal length to positively identify VH        genes.    -   Sequences without two canonical cysteines were rejected as these        were used to identify VH genes and reading frames.    -   Frameworks 1 to 4 were searched for based on homology with VH        genes in a database. If one or more of these frameworks were not        found, the sequence was rejected.    -   CDR1, 2 and 3 were identified based on the identified framework        regions. The sequence was rejected when a stop codon was present        in one or more of the CDRs.

Sequences that passed these criteria were classified as annotated VHsequences. All selected VH sequences were then submitted to anotheralgorithm to group them into clusters with a 100% identical HCDR3.

These data resulted in annotated VH genes and these VH genes wereclustered based on HCDR3 sequence. To analyze the selective expansion ofHCDR3 regions in VH genes in immunized versus non-immunized mice, theresults were tabulated and expressed as the ratio of annotated VHregions over clusters with identical HCDR3 regions (Table 15). For easeof interpretation, results for separate B cell fractions fromnon-immunized mice were pooled so that the ratio could analyzed fortotal mature, antigen-naïve B cells.

TABLE 15 Deep sequencing data from immunized versus non-immunized mice.Clusters Analyzed with Ratio B cell Annotated identical VH/ Immunizationpopulation VH regions HCDR3 clusters None Total mature B 39,626 30,7161.3 cells* None Total mature B 6,102 5,676 1.1 cells* None Total matureB 9,128 8,437 1.1 cells* cMet protein Total B cells 34,327 2,757 12.5cMet protein Total B cells 90,049 4,511 20.0 cMet protein & Total Bcells 19,645 3,233 6.1 cells cMet protein & Total B cells 75,838 4,55716.6 cells EGFR protein IgG⁺ B cells 46,924 4518 10.4 EGFR protein IgG⁺B cells 3,799 1201 3.2 EGFR protein & Total B cells 60,979 3526 17.3cells EGFR protein & Total B cells 43,631 2452 17.8 cells *B cellsexpressing either IgM or IgD.

From table 15 it can readily be observed that the number of clusterswith identical HCDR3 in non-immunized mice is in the order of the numberof annotated VH genes, which is reflected in VH/cluster ratios near 1.0.This implies that in these mice there was no trigger for selectiveexpansion of B cells that would carry VHs with certain HCDR3 regions, aswould be expected in the absence of an immunogenic stimulus. Incontrast, in immunized mice the ratio of VH regions over clusters withidentical HCDR3 is, although variable between individual mice, muchhigher and VH/cluster ratios range from 3.2 to 20.0. Thus, in immunizedmice a large fraction of HCDR3 sequences is present in high frequency inthe repertoire probably as a result of clonal expansion ofantigen-reactive B cells due to immunization of the mice. This indicatesthat the high frequency VH regions are likely the clones that arespecific for the antigen of interest and suggests that mining these VHgenes and expressing them as Fab or IgG together with the common lightchain will likely render antibodies with specificity for the antigen.Data that this is indeed the case are shown in Example 9. Functionalityof these antigen-specific antibodies can subsequently be tested infunctional assays.

Example 9: Deep Sequencing of VH Repertoires and HCDR3 Heat MapGeneration from Immunized Mice Expressing a Common Light Chain

As is clear from Example 8, a large fraction of HCDR3 sequences ispresent at a high frequency in the VH repertoire of immunized micecarrying a common light chain as opposed to non-immunized mice. In thepresent example, the repertoire of these most frequently used VH genesfrom immunized mice was mined to find antigen-specific heavychain-derived binding domains which upon combination with the commonlight chain will render functional antibodies against the target ofinterest.

Material from mice carrying the huVκ1-39 transgene and a human HCminilocus upon immunization with cMet or EGFR (protein-Fc, or protein-Fcalternating with cells expressing the respective proteins at theirsurface; two mice per strategy so eight in total) was generated andprocessed as described in Example 8.

Deep sequencing and VH repertoire analysis including clustering based onHCDR3 identity revealed a total 287920 sequences which could be groupedinto 813 unique clusters. All sequences in a cluster were derived fromthe same germline VH gene. Clusters that contained more than 50 VHs(with identical HCDR3 at the amino acid level but otherwise different VHregions) were collected into one database for repertoires from all eightmice. This was done since (limited) overlap of HCDR3 sequences wasobserved between the repertoires of the eight mice. VH sequences werethen ranked on HCDR3 length and HCDR3 sequence identity. Next, HCDR3sequences were further grouped based on the likelihood of a unique VDJ(i.e. if HCDR3 in different clusters contained <2 amino acids differencethen they were considered part of the same cluster and were groupedtogether). This process was performed manually in an Excel worksheet butcould be better performed on the basis of HCDR3 nucleotide sequencealignment. Tools to facilitate this are in development. This resulted ina total of 399 clusters with the same number of total sequences. Theclusters were then aligned based on their size (i.e. total number ofsequences in the cluster) and the top ˜100 clusters for each targetselected. This resulted in a total of 228 unique sequences: 134 fromcMet-immunized mice and 94 from EGFR-immunized mice. Finally to selectthe nucleotide sequence to be recloned as an IgG from each cluster, theVH amino acid sequence alignment for each cluster was analyzed and themost frequent sequence in the cluster was chosen for recloning.

The corresponding nucleotide sequences were retrieved, modified tocontain restriction sites for cloning into an expression vector and forremoval of excessive restriction sites by silent mutation and thensynthesized according to procedures known to the skilled person.Synthesized genes were cloned in bulk into a vector for expression inhuman IgG1 format including the huVκ1-39 common light chain. Of therendered clones, 400 clones were picked by standard procedures andsequenced, and bulk cloning was repeated for missing or incorrectsequences until >90% of the 228-repertoire was retrieved. DNA wasrecovered and transiently transfected into an antibody production cellline. Methods used to modify sequences, synthesize DNA, clone, sequenceand produce IgG by transfection are all known in the art.

IgG concentration of productions was determined using Octet technology(FortéBIO) according to the manufacturer's instructions for basicquantitation with a protein A sensor chip with regeneration (FortéBIO,cat. no. 18-0004/-5010/-5012/-5013). The 228 IgG were subsequentlysubmitted to testing for antigen specific binding by ELISA at aconcentration of 5 μg/ml. Of the tested sequences, 16 out of 110 (15%)were found to specifically bind to cMet and 6 out of 88 (7%) to EGFR.The other frequent clusters represent, for the large majority,antibodies that are directed to the Fc part of the antigen. It should benoted that the Fc-tail of the Fc-fusion proteins can be particularlyimmunogenic (Ling 1987, Immunology, vol 62, part 1, pp 1-6). Since thesesequences were derived from material of mice immunized with Fc-fusionproteins in adjuvant, a large part of the humoral response will bedirected to the Fc-tail (see Example 3 and 4). Binding to cellsexpressing the antigen on their surface was only done for IgG withbinding domains derived from mice that were immunized with protein andcells, using cMet-expressing MKN45 cells or EGFR-expressing A431 cells.All IgG from cell-immunized mice that stained cMet or EGFR in ELISA alsostained cells expressing the respective antigens (data not shown. It isexpected that upon immunization with pure antigen such as via DNAtattoo, the percentage of antigen-specific clones will be furtherincreased.

To functionally characterize the EGFR-specific IgG, these were testedfor their potency to inhibit EGF-induced cell death of A431 cells (Gulliet al. 1996, Cell Growth Diff 7, p. 173-178). Of the 5 tested anti-EGFRmAbs, 3 were shown to inhibit EGF-induced A431 cell death (FIG. 10). ThecMet-specific IgG were tested for functionality by determining theircapacity to compete with several benchmark antibodies obtained from(patent) literature for binding to cMet in a binding competition ELISA.Briefly, 96-wells plates were coated with cMet-Fc and then incubatedwith an excess of one of several bench mark antibodies. Subsequently,tagged cMet-specific binders were added, followed by aperoxydase-labeled detection antibody that recognized bound cMetbinders. The latter was detected using TMB as a substrate. Of the 10tested cMet mAbs, 7 were demonstrated to compete for cMet binding withbench mark antibodies.

Thus, by using deep sequencing methods a broad panel of diverseantigen-specific VHs can be identified representing diverse VH usage,diverse HCDR3, and clonal maturation.

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The invention claimed is:
 1. A method for producing a population ofantibodies which bind to an antigen, said method comprising: a)providing a population of B cells from a transgenic mouse immunized withsaid antigen; wherein said transgenic mouse expresses a rearranged humanantibody light chain variable region (VL) from a VL nucleic acid,wherein said transgenic mouse expresses a human antibody heavy chainvariable region (VH) repertoire from a repertoire of VH nucleic acids,said B cell population expressing a repertoire of VHs and a limitedimmunoglobulin light chain variable (VL) region repertoire, b) obtainingthe nucleic acids encoding antibody heavy chain variable (VH) regionsfrom said population of B cells, c) obtaining nucleotide sequences ofthe obtained nucleic acids of step b), d) determining the frequency ofnucleotide sequences from step c) which encode VH regions, e) providinghost cells, wherein each host cell comprises at least one vectorcomprising a nucleotide sequence encoding at least one VH region havinga sequence from those determined in step d) wherein each said host cellexpresses the rearranged human VL nucleic acid of step a) and said atleast one VH region, f) culturing said host cells from step e) andallowing for expression of said VL and VH regions, and g) obtaining saidpopulation of antibodies from the cultured host cells of step f) whichbind said antigen, wherein the method is without performing hybridomageneration or use of display technology between steps b) and c).
 2. Themethod of claim 1, further comprising amplifying nucleic acids encodingantibody heavy chain variable regions obtained in step b).
 3. The methodof claim 1, further comprising subjecting a sample of said cultured hostcells of step g) to at least one functional assay to confirm binding tosaid antigen, and selecting at least one cell that produces an antibodywhich binds said antigen.
 4. The method of claim 1, wherein thetransgenic mouse has been immunized with a nucleic acid encoding saidantigen or with a protein form of said antigen.
 5. The method of claim1, wherein the host cells of step e) further contain at least one secondnucleic acid for expression of nucleotide sequence that encodes a secondVH region that binds to a different antigen from that bound by the firstVH region and said population of antibodies in step g) comprisesbispecific antibodies.
 6. The method of claim 5, wherein said secondnucleic acid encoding the second VH region is identified by: a)providing a population of B cells from a transgenic mouse immunized withsaid antigen; wherein said transgenic mouse expresses a rearranged humanantibody light chain variable region (VL) from a VL nucleic acid,wherein said transgenic mouse expresses a human antibody heavy chainvariable region (VH) repertoire from a repertoire of VH nucleic acids,said B cell population expressing a repertoire of VHs and the rearrangedhuman antibody VL, said B cell population thus expressing a limitedimmunoglobulin light chain variable (VL) region repertoire, b) obtainingthe nucleic acids encoding antibody heavy chain variable (VH) regionsfrom said population of B cells, c) obtaining nucleotide sequences ofthe obtained nucleic acids of step b), d) determining the frequency ofnucleotide sequences from step c) which encode VH regions, e) selectingnucleic acids encoding VH regions comprising frequently occurring HCDR3amino acid sequences identified in step d).
 7. The method of claim 6,wherein the transgenic mouse has been immunized with a nucleic acidencoding said antigen or with a protein form of said antigen.